Antibodies and methods of use thereof

ABSTRACT

The present disclosure provides multispecific (e.g., bispecific) antibodies that specifically bind to human GITR and/or human OX40 as well as compositions comprising such antibodies. In a specific aspect, the multispecific antibodies specifically bind to human GITR and OX40 and modulate GITR and/or OX40 activity, e.g., enhance, activate, or induce GITR and/or OX40 activity, or reduce, deactivate, or inhibit GITR and/or OX40 activity. The present disclosure also provides methods for treating disorders, such as cancer, by administering a multispecific antibody that specifically binds to human GITR and/or OX40 and modulates GITR and/or OX40 activity, e.g., enhances, activates, or induces GITR and/or OX40 activity. Also provided are methods for treating autoimmune or inflammatory diseases or disorders, by administering a multispecific antibody that specifically binds to human GITR and/or OX40 and modulates GITR and/or OX40 activity, e.g., reduces, deactivates, or inhibits GITR and/or OX40 activity.

1. RELATED APPLICATIONS

This Application is a Division of U.S. application Ser. No. 15/781,043,filed on Jun. 1, 2018, issued as U.S. Pat. No. 11,447,557 on Sep. 20,2022 which was was the National Stage of Intenrational Application No.PCT/US2016/064642, filed on Dec. 2, 2016, which claims priority to U.S.Provisional Application No. 62/262,369, filed on Dec. 2, 2015, and62/419,911, filed on Nov. 9, 2016, the disclosures of which are hereinincorporated by reference in their entireties.

2. SEQUENCE LISTING

The instant application contains a sequence listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety (said XML copy, created on Jun. 5, 2023, isnamed 193050_SL.xml and is 201,601 bytes in size).

3. FIELD

The present disclosure relates to multispecific antibodies, e.g.,bispecific antibodies, that specifically bind to humanglucocorticoid-induced TNFR family related receptor (GITR) and/or humanOX40 receptor (“OX40”), compositions comprising such antibodies, andmethods of producing and using those antibodies.

4. BACKGROUND

The contributions of the innate and adaptive immune response in thecontrol of human tumor growth are well-characterized (Vesely M D et al.,(2011) Annu Rev Immunol 29: 235-271). As a result, antibody-basedstrategies have emerged that aim to enhance T cell responses for thepurpose of cancer therapy, such as targeting T cell expressedstimulatory receptors with agonist antibodies, or inhibitory receptorswith functional antagonists (Mellman I et al., (2011) Nature 480:480-489). Antibody-mediated agonist and antagonist approaches have shownpreclinical, and more recently clinical, activity.

Two important stimulators of immune responses are glucocorticoid-inducedTNFR-related protein (GITR) and OX40 receptor (“OX40”). Both GITR andOX40 are members of the tumor necrosis factor receptor superfamily(TNFRSF).

GITR (also known as activation-inducible TNFR family receptor (AITR),GITR-D, CD357, and tumor necrosis factor receptor superfamily member 18(TNFRSF18)), is expressed in many components of the innate and adaptiveimmune system and stimulates both acquired and innate immunity(Nocentini G et al., (1994) PNAS 94: 6216-6221; Hanabuchi S et al.,(2006) Blood 107:3617-3623; Nocentini G & Riccardi C (2005) Eur JImmunol 35: 1016-1022; Nocentini G et al., (2007) Eur J Immunol37:1165-1169). It is expressed in several cells and tissues, includingT, B, dendritic (DC) and Natural Killer (NK) cells and is activated byits ligand, GITRL, mainly expressed on Antigen Presenting Cells (APCs),on endothelial cells, and also in tumor cells. The GITR/GITRL systemparticipates in the development of autoimmune/inflammatory responses andpotentiates response to infection and tumors. For example, treatinganimals with GITR-Fc fusion protein ameliorates autoimmune/inflammatorydiseases while GITR triggering is effective in treating viral,bacterial, and parasitic infections, as well in boosting immune responseagainst tumors (Nocentini G et al., (2012) Br J Pharmacol 165: 2089-99).These effects are due to several concurrent mechanisms including:co-activation of effector T-cells, inhibition of regulatory T (Treg)cells, NK-cell co-activation, activation of macrophages, modulation ofdendritic cell function, and regulation of the extravasation process.The membrane expression of GITR is increased following T cell activation(Hanabuchi S et al., (2006) supra; Nocentini G & Riccardi C supra). Itstriggering coactivates effector T lymphocytes (McHugh R S et al., (2002)Immunity 16: 311-323; Shimizu J et al., (2002) Nat Immunol 3: 135-142;Roncheti S et al., (2004) Eur J Immunol 34: 613-622; Tone M et al.,(2003) PNAS 100: 15059-15064). GITR activation increases resistance totumors and viral infections, is involved in autoimmune/inflammatoryprocesses and regulates leukocyte extravasation (Nocentini G & RiccardiC (2005) supra; Cuzzocrea S et al., (2004) J Leukoc Biol 76: 933-940;Shevach E M & Stephens G L (2006) Nat Rev Immunol 6: 613-618; CuzzocreaS et al., (2006) J Immunol 177: 631-641; Cuzzocrea S et al., (2007)FASEB J 21: 117-129).

Human GITR is expressed at very low levels in peripheral (non-activated)T cells. After T cell activation, GITR is strongly up-regulated forseveral days in both CD4⁺ and CD8⁺ cells (Kwon B et al., (1999) J BiolChem 274: 6056-6061; Gurney A L et al., (1999) Curr Biol 9: 215-218;Ronchetti S et al., (2004) supra; Shimizu J et al., (2002) supra; Ji H Bet al., (2004) supra; Ronchetti S et al., (2002) Blood 100: 350-352; LiZ et al., (2003) J Autoimmun 21: 83-92), with CD4⁺ cells having a higherGITR expression than CD8⁺ cells (Kober J et al., (2008) Eur J Immunol38(10): 2678-88; Bianchini R et al., (2011) Eur J Immunol 41(8):2269-78) OX40 (also known as CD134, tumor necrosis factor receptorsuperfamily member 4 (TNFRSF4), TXGP1L, ACT35, and ACT-4) modulates Tcell, Natural Killer T (NKT) cell, and NK cell function (Sugamura K etal., (2004) Nat Rev Immunol 4: 420-431). OX40 can be upregulated byantigen-specific T cells following T cell receptor (TCR) stimulation byprofessional antigen presenting cells (APCs) displaying MHC class I orII molecules loaded with a cognate peptide (Sugamura K et al., (2004)Nat Rev Immunol 4: 420-431). Upon maturation APCs such as dendriticcells (DCs) upregulate stimulatory B7 family members (e.g., CD80 andCD86), as well as accessory co-stimulatory molecules including OX40ligand (OX40L), which help to sculpt the kinetics and magnitude of the Tcell immune response, as well as effective memory cell differentiation.Notably, other cell types can also express constitutive and/or induciblelevels of OX40L such as B cells, vascular endothelial cells, mast cells,and in some instances activated T cells (Soroosh P et al., (2006) JImmunol 176: 5975-5987). OX40:OX40L co-engagement is believed to drivethe higher order clustering of receptor trimers and subsequent signaltransduction (Compaan D M et al., (2006) Structure 14: 1321-1330).

OX40 expression by T cells within the tumor microenvironment has beenobserved in murine and human tumor tissues (Bulliard Y et al., (2014)Immunol Cell Biol 92: 475-480 and Piconese S et al., (2014) Hepatology60: 1494-1507). OX40 is highly expressed by intratumoral populations ofregulatory T cells (Tregs) relative to conventional T cell populations,a feature attributed to their proliferative status (Waight J D et al.,(2015) J Immunol 194: 878-882 and Bulliard Y et al., (2014) Immunol CellBiol 92: 475-480). Early studies demonstrated that OX40 agonistantibodies were able to elicit tumor rejection in mouse models (WeinbergA D et al., (2000) J Immunol 164: 2160-2169 and Piconese S et al.,(2008) J Exp Med 205: 825-839). A mouse antibody that agonizes humanOX40 signaling has also been shown to enhance immune functions in cancerpatients (Curti B D et al., (2013) Cancer Res 73: 7189-7198).

OX40 and OX40L interactions also have been associated with immuneresponses in inflammatory and autoimmune diseases and disorders,including mouse models of asthma/atopy, encephalomyelitis, rheumatoidarthritis, colitis/inflammatory bowel disease, graft-versus-host disease(e.g., transplant rejection), diabetes in non-obese diabetic mice, andatherosclerosis (Croft M et al., (2009) Immunol Rev 229(1): 173-191, andreferences cited therein). Reduced symptomatology associated with thediseases and disorders has been reported in OX40- and OX40L-deficientmice, in mice receiving anti-OX40 liposomes loaded with a cytostaticdrug, and in mice in which OX40 and OX40L interactions were blocked withan anti-OX40L blocking antibody or a recombinant OX40 fused to the Fcportion of human immunoglobulin (Croft M et al.; Boot E P J et al.,(2005) Arthritis Res Ther 7: R604-615; Weinberg A D et al., (1999) JImmunol 162: 1818-1826). Treatment with a blocking anti-OX40L antibodywas also shown to inhibit Th2 inflammation in a rhesus monkey model ofasthma (Croft M et al., Seshasayee D et al., (2007) J Clin Invest 117:3868-3878). Additionally, polymorphisms in OX40L have been associatedwith lupus (Croft M et al.)

Given the role of human GITR and OX40 in modulating immune responses,provided herein are antibodies that specifically bind to GITR and/orOX40 and the use of these antibodies to modulate GITR and/or OX40activity.

5. SUMMARY

In one aspect, provided herein are multispecific (e.g., bispecific)antibodies that bind to GITR and/or OX40. In one instance, an isolatedmultispecific (e.g., bispecific) antibody comprises a firstantigen-binding domain that specifically binds to OX40 (e.g., humanOX40) and a second antigen-binding domain that specifically binds toGITR (e.g., human GITR).

In one instance, an antibody comprises a first antigen-binding domainthat specifically binds to human OX40 and a TNF superfamily protein. Inone instance, an antibody comprises a TNF superfamily protein and asecond antigen-binding domain that specifically binds to human GITR. ATNF superfamily protein can replace the first antigen-binding domain orthe second antigen-binding domain in any multispecific (e.g.,bispecific) antibody provided herein.

In one instance, an isolated multispecific (e.g., bispecific) antibodythat specifically binds to human OX40 comprises: (a) a firstantigen-binding domain that specifically binds to human OX40;comprising: (i) a heavy chain variable domain (VH)-complementaritydetermining region (CDR) 1 comprising the amino acid sequence of GSAMH(SEQ ID NO:47); (ii) a VH-CDR2 comprising the amino acid sequence ofRIRSKANSYATAYAASVKG (SEQ ID NO:48); (iii) a VH-CDR3 comprising the aminoacid sequence of GIYDSSGYDY (SEQ ID NO:49); (iv) a light chain variabledomain (VL)-CDR1 comprising the amino acid sequence of RSSQSLLHSNGYNYLD(SEQ ID NO:50); (v) a VL-CDR2 comprising the amino acid sequence ofLGSNRAS (SEQ ID NO:51); and (vi) a VL-CDR3 comprising the amino acidsequence of MQGSKWPLT (SEQ ID NO:52) or MQALQTPLT (SEQ ID NO:53); and(b) a second antigen-binding domain.

In one instance, an isolated multispecific (e.g., bispecific) antibodythat specifically binds to human OX40 comprises: (a) a firstantigen-binding domain that specifically binds to the same epitope ofhuman OX40 as an antibody comprising a VH comprising the amino acidsequence of SEQ ID NO:54 and a VL comprising the amino acid sequence ofSEQ ID NO:55 or 56; and (b) a second antigen-binding domain.

In one instance an isolated multispecific (e.g., bispecific) antibodythat specifically binds to human OX40 comprises: (a) a firstantigen-binding domain that specifically binds to human OX40 andexhibits, as compared to binding to a human OX40 sequence of SEQ IDNO:72, reduced or absent binding to a protein identical to SEQ ID NO:72except for the presence of an amino acid mutation selected from thegroup consisting of: N60A, R62A, R80A, L88A, P93A, P99A, P115A, and acombination thereof, numbered according to SEQ ID NO: 72; and (b) asecond antigen-binding domain.

In one instance, an isolated multispecific (e.g., bispecific) antibodythat specifically binds to human OX40 comprises: (a) a firstantigen-binding domain that specifically binds to human OX40 comprisinga VH and a VL, wherein the VH comprises the amino acid sequence of SEQID NO:54; and (b) a second antigen-binding domain.

In one instance, an isolated multispecific (e.g., bispecific) antibodythat specifically binds to human OX40 comprises: (a) a firstantigen-binding domain that specifically binds to human OX40 comprisinga VH and a VL, wherein the VL comprises the amino acid sequence of SEQID NO:55 or SEQ ID NO:56; (b) a second antigen-binding domain.

In one instance, an isolated multispecific (e.g., bispecific) antibodythat specifically binds to human GITR comprises: (a) a firstantigen-binding domain; and (b) a second antigen-binding domain thatspecifically binds to human GITR comprising (i) a VH-CDR1 comprising theamino acid sequence of X₁YX₂MX₃ (SEQ ID NO:87), wherein X₁ is D, E or G;X₂ is A or V; and X₃ is Y or H; (ii) a VH-CDR2 comprising the amino acidsequence of X₁IX₂TX₃SGX4X₅X₆YNQKFX₇X₈(SEQ ID NO:88), wherein X₁ is V orL; X₂ is R, K or Q; X₃ is Y or F; X₄ is D, E or G; X₅ is V or L; X₆ is Tor S; X₇ is K, R or Q; and X₈ is D, E or G; (iii) a VH-CDR3 comprisingthe amino acid sequence of SGTVRGFAY (SEQ ID NO:3); (iv) a VL-CDR1comprising the amino acid sequence of KSSQSLLNSX₁NQKNYLX₂ (SEQ IDNO:90), wherein X₁ is G or S; and X₂ is T or S; (v) a VL-CDR2 comprisingthe amino acid sequence of WASTRES (SEQ ID NO:5); and (vi) a VL-CDR3comprising the amino acid sequence of QNX₁YSX₂PYT (SEQ ID NO:92),wherein X₁ is D or E; and X₂ is Y, F or S.

In one instance, an isolated multispecific (e.g., bispecific) antibodythat specifically binds to human GITR comprises: (a) a firstantigen-binding domain; and (b) a second antigen-binding domain thatspecifically binds to the same epitope of human GITR as an antibodycomprising a VH comprising the amino acid sequence of SEQ ID NO:18 and aVL comprising the amino acid sequence of SEQ ID NO:19.

In one instance, an isolated multispecific (e.g., bispecific) antibodythat specifically binds to human GITR comprises: (a) a firstantigen-binding domain; and (b) a second antigen-binding domain thatspecifically binds to an epitope of human GITR comprising at least oneamino acid in residues 60-63 of SEQ ID NO:41.

In one instance, an isolated multispecific (e.g., bispecific) antibodythat specifically binds to human GITR comprises: (a) a firstantigen-binding domain; and (b) a second antigen-binding domain thatspecifically binds to each of i) human GITR comprising residues 26 to241 of SEQ ID NO:41 and ii) a variant of cynomolgus GITR, the variantcomprising residues 26-234 of SEQ ID NO:46, wherein the antibody doesnot specifically bind to cynomolgus GITR comprising residues 26-234 ofSEQ ID NO:44.

In one instance, an isolated multispecific (e.g., bispecific) antibodythat specifically binds to human GITR comprises: (a) a firstantigen-binding domain; and (b) a second antigen-binding domain thatspecifically binds to human GITR and exhibits, as compared to binding toa human GITR sequence of residues 26 to 241 of SEQ ID NO:41, reduced orabsent binding to a protein identical to residues 26 to 241 of SEQ IDNO:41 except for the presence of a D60A or G63A amino acid substitution,numbered according to SEQ ID NO:41.

In one instance, an isolated multispecific (e.g., bispecific) antibodythat specifically binds to human GITR comprises: (a) a firstantigen-binding domain; and (b) a second antigen-binding domain thatspecifically binds to human GITR and comprises a VH and a VL, whereinthe VH comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs:18, 20, 22, 24, and 25.

In one instance, an isolated multispecific (e.g., bispecific) antibodythat specifically binds to human GITR comprises: (a) a firstantigen-binding domain; and (b) a second antigen-binding domain thatspecifically binds to human GITR and comprises a VH and a VL, whereinthe VL comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs:19, 21, 23, and 26.

In one instance, the isolated multispecific (e.g., bispecific) antibodythat specifically binds to human OX40 comprises the secondantigen-binding domain. In one instance, the second antigen-bindingdomain specifically binds to a TNFR superfamily protein. In oneinstance, the TNFR superfamily protein is selected from the groupconsisting of: GITR, OX40 (e.g., wherein the second antigen-bindingdomain binds to a different OX40 epitope than the first antigen-bindingdomain), CD137, DR3, CD40, BAFFR, CD27, and HVEM.

In one instance, the isolated multispecific (e.g., bispecific) antibodythat specifically binds to human GITR comprises the firstantigen-binding domain. In one instance, the first antigen-bindingdomain specifically binds to a TNFR superfamily protein. In oneinstance, the TNFR superfamily protein is selected from the groupconsisting of: GITR (e.g., wherein the first antigen-binding domainbinds to a different GITR epitope than the second antigen-bindingdomain) OX40, CD137, DR3, CD40, BAFFR, CD27, and HVEM.

In one instance, the second antigen-binding domain specifically binds tothe TNFR superfamily protein human GITR. In one instance, the secondantigen-binding domain that binds to human GITR comprises: (i) a VH-CDR1comprising the amino acid sequence of X₁YX₂MX₃ (SEQ ID NO:87), whereinX₁ is D, E or G; X₂ is A or V; and X₃ is Y or H; (ii) a VH-CDR2comprising the amino acid sequence of X₁IX₂TX₃SGX₄X₅X₆YNQKFX₇X₈(SEQ IDNO:88), wherein X₁ is V or L; X₂ is R, K or Q; X₃ is Y or F; X₄ is D, Eor G; X₅ is V or L; X₆ is T or S; X₇ is K, R or Q; and X₈ is D, E or G;(iii) a VH-CDR3 comprising the amino acid sequence of SGTVRGFAY (SEQ IDNO:3); (iv) a VL-CDR1 comprising the amino acid sequence ofKSSQSLLNSX₁NQKNYLX₂ (SEQ ID NO:90), wherein X₁ is G or S; and X₂ is T orS; (v) a VL-CDR2 comprising the amino acid sequence of WASTRES (SEQ IDNO:5); and (vi) a VL-CDR3 comprising the amino acid sequence ofQNX₁YSX₂PYT (SEQ ID NO:92), wherein X₁ is D or E; and X₂ is Y, F or S.In one instance, the second antigen-binding domain that specificallybinds to human GITR binds to the same epitope of human GITR as anantibody comprising a VH comprising the amino acid sequence of SEQ IDNO:18 and a VL comprising the amino acid sequence of SEQ ID NO:19. Inone instance, the second antigen-binding domain that specifically bindsto human GITR binds to an epitope of human GITR comprising at least oneamino acid in residues 60-63 of SEQ ID NO:41. In one instance, thesecond antigen-binding domain that specifically binds to human GITRbinds to each of i) human GITR comprising residues 26 to 241 of SEQ IDNO:41 and ii) a variant of cynomolgus GITR, the variant comprisingresidues 26-234 of SEQ ID NO:46, wherein the second antigen-bindingdomain does not specifically bind to cynomolgus GITR comprising residues26-234 of SEQ ID NO:44. In one instance, the second antigen-bindingdomain that specifically binds to human GITR exhibits, as compared tobinding to a human GITR sequence of residues 26 to 241 of SEQ ID NO:41,reduced or absent binding to a protein identical to residues 26 to 241of SEQ ID NO:41 except for the presence of a D60A or G63A amino acidsubstitution, numbered according to SEQ ID NO:41. In one instance, thesecond antigen-binding domain that specifically binds to human GITRcomprises a VH and a VL, wherein the VH comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs:18, 20, 22, 24, and 25.In one instance, the second antigen-binding domain that specificallybinds to human GITR comprises a VH and a VL, wherein the VL comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:19,21, 23, and 26.

In one instance, the first antigen-binding domain specifically binds tothe TNFR superfamily protein human OX40. In one instance, the firstantigen-binding domain that specifically binds to human OX40 comprises:(i) a VH-CDR1 comprising the amino acid sequence of GSAMH (SEQ IDNO:47); (ii) a VH-CDR2 comprising the amino acid sequence ofRIRSKANSYATAYAASVKG (SEQ ID NO:48); (iii) a VH-CDR3 comprising the aminoacid sequence of GIYDSSGYDY (SEQ ID NO:49); (iv) a VL-CDR1 comprisingthe amino acid sequence of RSSQSLLHSNGYNYLD (SEQ ID NO:50); (v) aVL-CDR2 comprising the amino acid sequence of LGSNRAS (SEQ ID NO:51);and (vi) a VL-CDR3 comprising the amino acid sequence of MQGSKWPLT (SEQID NO:52) or MQALQTPLT (SEQ ID NO:53). In one instance, the firstantigen-binding domain that specifically binds to human OX40 binds tothe same epitope of human OX40 as an antibody comprising a VH comprisingthe amino acid sequence of SEQ ID NO:54 and a VL comprising the aminoacid sequence of SEQ ID NO:55 or 56. In one instance, the firstantigen-binding domain that specifically binds to human OX40 exhibits,as compared to binding to a human OX40 sequence of SEQ ID NO:72, reducedor absent binding to a protein identical to SEQ ID NO:72 except for thepresence of an amino acid mutation selected from the group consistingof: N60A, R62A, R80A, L88A, P93A, P99A, P115A, and a combinationthereof, numbered according to SEQ ID NO: 72. In one instance, the firstantigen-binding domain that specifically binds to human OX40 comprises aVH and a VL, and the VH comprises the amino acid sequence of SEQ IDNO:54. In one instance, the first antigen-binding domain thatspecifically binds to human OX40 comprises a VH and a VL, and the VLcomprises the amino acid sequence of SEQ ID NO:55 or SEQ ID NO:56.

In one instance, the second antigen-binding domain that specificallybinds to human GITR comprises: (i) a VH-CDR1 comprising the amino acidsequence of X₁YAMX₂ (SEQ ID NO:1), wherein X₁ is D, G, or E; and X₂ is Yor H; (ii) a VH-CDR2 comprising the amino acid sequence ofX₁IRTYSGX₂VX₃YNQKFX₄X₅ (SEQ ID NO:2), wherein X₁ is V or L; X₂ is D orG; X₃ is T or S; X₄ is K, R, or Q; and X₅ is D, E, or G; (iii) a VH-CDR3comprising the amino acid sequence of SGTVRGFAY (SEQ ID NO:3); (iv) aVL-CDR1 comprising the amino acid sequence of KSSQSLLNSX₁NQKNYLT (SEQ IDNO:4), wherein X₁ is G or S; (v) a VL-CDR2 comprising the amino acidsequence of WASTRES (SEQ ID NO:5); and (vi) a VL-CDR3 comprising theamino acid sequence of QNX₁YSX₂PYT (SEQ ID NO:6), wherein X₁ is D or E;and X₂ is Y or F.

In one instance, the second antigen-binding domain that specificallybinds to human GITR comprises a VH-CDR1 comprising an amino acidsequence selected from the group consisting of SEQ ID NOs:7-9. In oneinstance, the second antigen-binding domain that specifically binds tohuman GITR comprises a VH-CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs:10-13. In one instance,the second antigen-binding domain that specifically binds to human GITRcomprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:14or 15. In one instance, the second antigen-binding domain thatspecifically binds to human GITR comprises a VL-CDR3 comprising theamino acid sequence of SEQ ID NO:16 or 17.

In one instance, the second antigen-binding domain that specificallybinds to human GITR comprises VH-CDR1, VH-CDR2, and VH-CDR3 sequencesset forth in SEQ ID NOs:7, 10, and 3; SEQ ID NOs:8, 11, and 3; SEQ IDNOs:9, 12, and 3; or SEQ ID NOs:9, 13, and 3, respectively; and/orVL-CDR1, VL-CDR2, and VL-CDR3 sequences set forth in SEQ ID NOs:14, 5,and 16; or SEQ ID NOs:15, 5, and 17, respectively.

In one instance, the second antigen-binding domain that specificallybinds to human GITR comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1,VL-CDR2, and VL-CDR3 sequences set forth in SEQ ID NOs:7, 10, 3, 14, 5,and 16, respectively.

In one instance, the second antigen-binding domain that specificallybinds to human GITR comprises a VH comprising the amino acid sequenceset forth in SEQ ID NO:25. In one instance, the second antigen-bindingdomain that specifically binds to human GITR comprises a VH comprisingan amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99%identical to an amino acid sequence selected from the group consistingof SEQ ID NOs:18, 20, 22, and 24. In one instance, the secondantigen-binding domain that specifically binds to human GITR comprises aVH comprising an amino acid sequence selected from the group consistingof SEQ ID NOs:18, 20, 22, and 24. In one instance, the secondantigen-binding domain that specifically binds to human GITR comprises aVH comprising the amino acid sequence of SEQ ID NO:18.

In one instance, the second antigen-binding domain that specificallybinds to human GITR comprises a heavy chain comprising an amino acidsequence selected from the group consisting of SEQ ID NOs:29-36. In oneinstance, the second antigen-binding domain that specifically binds tohuman GITR comprises a heavy chain comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs:74-81. In one instance,the second antigen-binding domain that specifically binds to human GITRcomprises a heavy chain comprising the amino acid sequence of SEQ IDNO:31. In one instance, the second antigen-binding domain thatspecifically binds to human GITR comprises a heavy chain comprising theamino acid sequence of SEQ ID NO:76. In one instance, the secondantigen-binding domain that specifically binds to human GITR comprises aheavy chain comprising the amino acid sequence of SEQ ID NO:32. In oneinstance, the second antigen-binding domain that specifically binds tohuman GITR comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO:77. In one instance, the second antigen-binding domain thatspecifically binds to human GITR comprises a heavy chain comprising theamino acid sequence of SEQ ID NO:34. In one instance, the secondantigen-binding domain that specifically binds to human GITR comprises aheavy chain comprising the amino acid sequence of SEQ ID NO:79. In oneinstance, the second antigen-binding domain that specifically binds tohuman GITR comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO:35. In one instance, the second antigen-binding domain thatspecifically binds to human GITR comprises a heavy chain comprising theamino acid sequence of SEQ ID NO:80.

In one instance, the second antigen-binding domain that specificallybinds to human GITR comprises a VH comprising an amino acid sequencederived from a human IGHV1-2 germline sequence.

In one instance, the second antigen-binding domain that specificallybinds to human GITR comprises a VL comprising the amino acid sequence ofSEQ ID NO:26. In one instance, the second antigen-binding domain thatspecifically binds to human GITR comprises a VL comprising an amino acidsequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to an aminoacid sequence selected from the group consisting of SEQ ID NOs:19, 21,and 23. In one instance, the second antigen-binding domain thatspecifically binds to human GITR comprises a VL comprising an amino acidsequence selected from the group consisting of SEQ ID NOs:19, 21, and23. In one instance, the second antigen-binding domain that specificallybinds to human GITR comprises a VL comprising the amino acid sequence ofSEQ ID NO:19.

In one instance, the second antigen-binding domain that specificallybinds to human GITR comprises a light chain comprising the amino acidsequence of SEQ ID NO:37. In one instance, the second antigen-bindingdomain that specifically binds to human GITR comprises a light chaincomprising the amino acid sequence of SEQ ID NO:38.

In one instance, the second antigen-binding domain that specificallybinds to human GITR comprises a VL comprising an amino acid sequencederived from a human IGKV4-1 germline sequence.

In one instance, the second antigen-binding domain that specificallybinds to human GITR comprises VH and VL sequences set forth in SEQ IDNOs:18 and 19, SEQ ID NOs:20 and 21, SEQ ID NOs:22 and 23, or SEQ IDNOs:24 and 23, respectively. In one instance, the second antigen-bindingdomain that specifically binds to human GITR comprises a VH comprisingthe sequence set forth in SEQ ID NO:18 and a VL comprising the sequenceset forth in SEQ ID NO:19.

In one instance, the second antigen-binding domain that specificallybinds to human GITR comprises a heavy chain comprising the amino acidsequence of SEQ ID NO:31 and a light chain comprising the amino acidsequence of SEQ ID NO:37. In one instance, the second antigen-bindingdomain that specifically binds to human GITR comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:76 and a light chaincomprising the amino acid sequence of SEQ ID NO:37. In one instance, thesecond antigen-binding domain that specifically binds to human GITRcomprises a heavy chain comprising the amino acid sequence of SEQ IDNO:32 and a light chain comprising the amino acid sequence of SEQ IDNO:37. In one instance, the second antigen-binding domain thatspecifically binds to human GITR comprises a heavy chain comprising theamino acid sequence of SEQ ID NO:77 and a light chain comprising theamino acid sequence of SEQ ID NO:37. In one instance, the secondantigen-binding domain that specifically binds to human GITR comprises aheavy chain comprising the amino acid sequence of SEQ ID NO:34 and alight chain comprising the amino acid sequence of SEQ ID NO:37. In oneinstance, the second antigen-binding domain that specifically binds tohuman GITR comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO:79 and a light chain comprising the amino acid sequence of SEQID NO:37. In one instance, the second antigen-binding domain thatspecifically binds to human GITR comprises a heavy chain comprising theamino acid sequence of SEQ ID NO:35 and a light chain comprising theamino acid sequence of SEQ ID NO:37. In one instance, the secondantigen-binding domain that specifically binds to human GITR comprises aheavy chain comprising the amino acid sequence of SEQ ID NO:80 and alight chain comprising the amino acid sequence of SEQ ID NO:37. In oneinstance, the second antigen-binding domain that specifically binds tohuman GITR comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO:29 and a light chain comprising the amino acid sequence of SEQID NO:37. In one instance, the second antigen-binding domain thatspecifically binds to human GITR comprises a heavy chain comprising theamino acid sequence of SEQ ID NO:74 and a light chain comprising theamino acid sequence of SEQ ID NO:37. In one instance, the secondantigen-binding domain that specifically binds to human GITR comprises aheavy chain comprising the amino acid sequence of SEQ ID NO:30 and alight chain comprising the amino acid sequence of SEQ ID NO:37. In oneinstance, the second antigen-binding domain that specifically binds tohuman GITR comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO:75 and a light chain comprising the amino acid sequence of SEQID NO:37.

In one instance, the second antigen-binding domain that specificallybinds to human GITR comprises a heavy chain variable region having anamino acid sequence derived from a human IGHV1-2 germline sequence and alight chain variable region having an amino acid sequence derived from ahuman IGKV4-1 germline sequence.

In one instance, the first antigen-binding domain that specificallybinds to human OX40 comprises a VH comprising an amino acid sequencethat is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to the aminoacid sequence of SEQ ID NO:54. In one instance, the firstantigen-binding domain that specifically binds to human OX40 comprises aVH comprising the amino acid sequence of SEQ ID NO:54.

In one instance, the first antigen-binding domain that specificallybinds to human OX40 comprises a heavy chain comprising an amino acidsequence selected from the group consisting of SEQ ID NOs:59-66. In oneinstance, the first antigen-binding domain that specifically binds tohuman OX40 comprises a heavy chain comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs:118-125. In oneinstance, the first antigen-binding domain that specifically binds tohuman OX40 comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO:61. In one instance, the first antigen-binding domain thatspecifically binds to human OX40 comprises a heavy chain comprising theamino acid sequence of SEQ ID NO:120. In one instance, the firstantigen-binding domain that specifically binds to human OX40 comprises aheavy chain comprising the amino acid sequence of SEQ ID NO:62. In oneinstance, the first antigen-binding domain that specifically binds tohuman OX40 comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO:121. In one instance, the first antigen-binding domain thatspecifically binds to human OX40 comprises a heavy chain comprising theamino acid sequence of SEQ ID NO:64. In one instance, the firstantigen-binding domain that specifically binds to human OX40 comprises aheavy chain comprising the amino acid sequence of SEQ ID NO:123. In oneinstance, the first antigen-binding domain that specifically binds tohuman OX40 comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO:65. In one instance, the first antigen-binding domain thatspecifically binds to human OX40 comprises a heavy chain comprising theamino acid sequence of SEQ ID NO:124.

In one instance, the first antigen-binding domain that specificallybinds to human OX40 comprises a VH comprising an amino acid sequencederived from a human IGHV3-73 germline sequence.

In one instance, the first antigen-binding domain that specificallybinds to human OX40 comprises a VL comprising an amino acid sequence atleast 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acidsequence of SEQ ID NO:55 or SEQ ID NO:56. In one instance, the firstantigen-binding domain that specifically binds to human OX40 comprises aVL-CDR3 comprising the amino acid sequence of SEQ ID NO:52. In oneinstance, the first antigen-binding domain that specifically binds tohuman OX40 comprises a VL comprising the amino acid sequence of SEQ IDNO:55. In one instance, the first antigen-binding domain thatspecifically binds to human OX40 comprises a light chain comprising theamino acid sequence of SEQ ID NO:67. In one instance, the firstantigen-binding domain that specifically binds to human OX40 comprises alight chain comprising the amino acid sequence of SEQ ID NO:68.

In one instance, the first antigen-binding domain that specificallybinds to human OX40 comprises a VL-CDR3 comprising the amino acidsequence of SEQ ID NO:53. In one instance, the first antigen-bindingdomain that specifically binds to human OX40 comprises a VL comprisingthe amino acid sequence of SEQ ID NO:56. In one instance, the firstantigen-binding domain that specifically binds to human OX40 comprises alight chain comprising the amino acid sequence of SEQ ID NO:69. In oneinstance, the first antigen-binding domain that specifically binds tohuman OX40 comprises a light chain comprising the amino acid sequence ofSEQ ID NO:70.

In one instance, the first antigen-binding domain that specificallybinds to human OX40 comprises a VL comprising an amino acid sequencederived from a human IGKV2-28 germline sequence.

In one instance, the first antigen-binding domain that specificallybinds to human OX40 comprises the VH and VL sequences set forth in SEQID NOs:54 and 55 or SEQ ID NOs:54 and 56, respectively. In one instance,the first antigen-binding domain that specifically binds to human OX40comprises a heavy chain comprising the amino acid sequence of SEQ IDNO:59 and a light chain comprising the amino acid sequence of SEQ IDNO:67 or 69. In one instance, the first antigen-binding domain thatspecifically binds to human OX40 comprises a heavy chain comprising theamino acid sequence of SEQ ID NO:118 and a light chain comprising theamino acid sequence of SEQ ID NO:67 or 69. In one instance, the firstantigen-binding domain that specifically binds to human OX40 comprises aheavy chain comprising the amino acid sequence of SEQ ID NO:64 and alight chain comprising the amino acid sequence of SEQ ID NO:67 or 69. Inone instance, the first antigen-binding domain that specifically bindsto human OX40 comprises a heavy chain comprising the amino acid sequenceof SEQ ID NO:123 and a light chain comprising the amino acid sequence ofSEQ ID NO:67 or 69. In one instance, the first antigen-binding domainthat specifically binds to human OX40 comprises a heavy chain comprisingthe amino acid sequence of SEQ ID NO:65 and a light chain comprising theamino acid sequence of SEQ ID NO:67 or 69. In one instance, the firstantigen-binding domain that specifically binds to human OX40 comprises aheavy chain comprising the amino acid sequence of SEQ ID NO:124 and alight chain comprising the amino acid sequence of SEQ ID NO:67 or 69. Inone instance, the first antigen-binding domain that specifically bindsto human OX40 comprises a heavy chain comprising the amino acid sequenceof SEQ ID NO:61 and a light chain comprising the amino acid sequence ofSEQ ID NO:67 or 69. In one instance, the first antigen-binding domainthat specifically binds to human OX40 comprises a heavy chain comprisingthe amino acid sequence of SEQ ID NO:120 and a light chain comprisingthe amino acid sequence of SEQ ID NO:67 or 69. In one instance, thefirst antigen-binding domain that specifically binds to human OX40comprises a heavy chain comprising the amino acid sequence of SEQ IDNO:62 and a light chain comprising the amino acid sequence of SEQ IDNO:67 or 69. In one instance, the first antigen-binding domain thatspecifically binds to human OX40 comprises a heavy chain comprising theamino acid sequence of SEQ ID NO:121 and a light chain comprising theamino acid sequence of SEQ ID NO:67 or 69.

In one instance, the first antigen-binding domain that specificallybinds to human OX40 comprises a VH comprising an amino acid sequencederived from a human IGHV3-73 germline sequence and a VL comprising anamino acid sequence derived from a human IGKV2-28 germline sequence.

In one instance, the first antigen-binding domain that specificallybinds to human OX40 comprises the VH sequence set forth in SEQ ID NO:54and the VL sequence set forth in SEQ ID NO:55 or 56, and the secondantigen-binding domain that specifically binds to human GITR comprisesthe VH sequence set forth in SEQ ID NO:18 and the VL sequence set forthin SEQ ID NO:19. In one instance, the first antigen-binding domain thatspecifically binds to human OX40 comprises the heavy chain sequence setforth in SEQ ID NO:59 and the light chain sequence set forth in SEQ IDNO:67 or 69, and the second antigen-binding domain that specificallybinds to human GITR comprises the heavy chain sequence set forth in SEQID NO:29 and the light chain sequence set forth in SEQ ID NO:37. In oneinstance, the first antigen-binding domain that specifically binds tohuman OX40 comprises the heavy chain sequence set forth in SEQ ID NO:118and the light chain sequence set forth in SEQ ID NO:67 or 69, andwherein the second antigen-binding domain that specifically binds tohuman GITR comprises the heavy chain sequence set forth in SEQ ID NO:74and the light chain sequence set forth in SEQ ID NO:37. In one instance,the first antigen-binding domain that specifically binds to human OX40comprises the heavy chain sequence set forth in SEQ ID NO:61 and thelight chain sequence set forth in SEQ ID NO:67 or 69, and the secondantigen-binding domain that specifically binds to human GITR comprisesthe heavy chain sequence set forth in SEQ ID NO:31 and the light chainsequence set forth in SEQ ID NO:37. In one instance, the firstantigen-binding domain that specifically binds to human OX40 comprisesthe heavy chain sequence set forth in SEQ ID NO:120 and the light chainsequence set forth in SEQ ID NO:67 or 69, and wherein the secondantigen-binding domain that specifically binds to human GITR comprisesthe heavy chain sequence set forth in SEQ ID NO:76 and the light chainsequence set forth in SEQ ID NO:37. In one instance, the firstantigen-binding domain that specifically binds to human OX40 comprisesthe heavy chain sequence set forth in SEQ ID NO:62 and the light chainsequence set forth in SEQ ID NO:67 or 69, and the second antigen-bindingdomain that specifically binds to human GITR comprises the heavy chainsequence set forth in SEQ ID NO:32 and the light chain sequence setforth in SEQ ID NO:37. In one instance, the first antigen-binding domainthat specifically binds to human OX40 comprises the heavy chain sequenceset forth in SEQ ID NO:121 and the light chain sequence set forth in SEQID NO:67 or 69, and wherein the second antigen-binding domain thatspecifically binds to human GITR comprises the heavy chain sequence setforth in SEQ ID NO:77 and the light chain sequence set forth in SEQ IDNO:37. In one instance, the first antigen-binding domain thatspecifically binds to human OX40 comprises the heavy chain sequence setforth in SEQ ID NO:64 and the light chain sequence set forth in SEQ IDNO:67 or 69, and the second antigen-binding domain that specificallybinds to human GITR comprises the heavy chain sequence set forth in SEQID NO:34 and the light chain sequence set forth in SEQ ID NO:37. In oneinstance, the first antigen-binding domain that specifically binds tohuman OX40 comprises the heavy chain sequence set forth in SEQ ID NO:123and the light chain sequence set forth in SEQ ID NO:67 or 69, andwherein the second antigen-binding domain that specifically binds tohuman GITR comprises the heavy chain sequence set forth in SEQ ID NO:79and the light chain sequence set forth in SEQ ID NO:37. In one instance,the first antigen-binding domain that specifically binds to human OX40comprises the heavy chain sequence set forth in SEQ ID NO:65 and thelight chain sequence set forth in SEQ ID NO:67 or 69, and the secondantigen-binding domain that specifically binds to human GITR comprisesthe heavy chain sequence set forth in SEQ ID NO:35 and the light chainsequence set forth in SEQ ID NO:37. In one instance, the firstantigen-binding domain that specifically binds to human OX40 comprisesthe heavy chain sequence set forth in SEQ ID NO:124 and the light chainsequence set forth in SEQ ID NO:67 or 69, and wherein the secondantigen-binding domain that specifically binds to human GITR comprisesthe heavy chain sequence set forth in SEQ ID NO:80 and the light chainsequence set forth in SEQ ID NO:37. In one instance, the firstantigen-binding domain that specifically binds to human OX40 comprisesthe light chain sequence set forth in SEQ ID NO:67. In one instance, thefirst antigen-binding domain that specifically binds to human OX40comprises the light chain sequence set forth in SEQ ID NO:69.

In one instance, the first antigen-binding domain that specificallybinds to human OX40 comprises a VH derived from a human IGHV3-73germline sequence and a VL derived from a human IGKV2-28 germlinesequence, and the second antigen-binding domain that specifically bindsto human GITR comprises a VH derived from a human IGHV1-2 germlinesequence and a VL derived from a human IGKV4-1 germline sequence.

In one instance, the antibody is a kappa-lambda body, a dual-affinityre-targeting molecule (DART), a knob-in-hole antibody, a strand-exchangeengineered domain body (SEEDbody) or a DuoBody® antibody (Genmab A/S).

In one instance, the first antigen-binding domain comprises a heavychain constant region selected from the group consisting of human IgG₁,human IgG₂, human IgG₃, human IgG₄, human IgA₁, and human IgA₂, and thesecond antigen-binding domain comprises a heavy chain constant regionselected from the group consisting of human IgG₁, human IgG₂, humanIgG₃, human IgG₄, human IgA₁, and human IgA₂. In one instance, the heavychain constant region of the first antigen-binding domain is human IgG₁,and the heavy chain constant region of the second antigen-binding domainis human IgG₁.

In one instance, the (a) the first antigen-binding domain comprises afirst heavy chain constant region comprising an F405L amino acidmutation; and (b) the second antigen-binding domain comprises a secondheavy chain constant region comprising a K409R amino acid mutation,numbered according to the EU numbering system. In one instance, (a) thefirst antigen-binding domain comprises a first heavy chain constantregion comprising a K409R amino acid mutation; and (b) the secondantigen-binding domain comprises a heavy chain constant regioncomprising an F405L amino acid mutation, numbered according to the EUnumbering system. In one instance, the heavy chain constant region ofthe first antigen-binding domain is an IgG₁. In one instance, the heavychain constant region of the second antigen-binding domain is an IgG₁.

In one instance, the first antigen-binding domain comprises a lightchain constant region selected from the group consisting of humanIgG_(κ) and IgG_(λ), and the second antigen-binding domain comprises alight chain constant region selected from the group consisting of humanIgG_(κ), and IgG_(λ).

In one instance, the second antigen-binding domain exhibits, as comparedto binding to a human GITR sequence of residues 26 to 241 of SEQ IDNO:41, reduced or absent binding to a protein identical to residues 26to 241 of SEQ ID NO:41 except for the presence of a D60A substitution,numbered according to SEQ ID NO:41. In one instance, the secondantigen-binding domain exhibits, as compared to binding to a human GITRsequence of residues 26 to 241 of SEQ ID NO:41, reduced or absentbinding to a protein identical to residues 26 to 241 of SEQ ID NO:41except for the presence of a G63A substitution, numbered according toSEQ ID NO: 41. In one instance, the second antigen-binding domain thatbinds to human GITR binds to at least one residue selected from thegroup consisting of residues 60, 62, and 63 of SEQ ID NO:41. In oneinstance, the second antigen-binding domain that binds to human GITRbinds to at least one residue selected from the group consisting ofresidues 62 and 63 of SEQ ID NO:41. In one instance, the secondantigen-binding domain that binds to human GITR binds to at least oneresidue selected from the group consisting of residues 60 and 63 of SEQID NO:41. In one instance, the second antigen-binding domain that bindsto human GITR binds to an epitope comprising residues 60-63 of SEQ IDNO:41.

In one instance, the antibody (i) shows increased binding to cellsexpressing human GITR and human OX40 as compared to a monospecificbivalent antibody that binds to human GITR and contains the same VH andVL as the second antigen-binding domain that binds to human GITR; and/or(ii) shows increased binding to cells expressing human GITR and humanOX40 as compared to a monospecific bivalent antibody that binds to humanOX40 and contains the same VH and VL as the first antigen-binding domainthat binds to human OX40.

In one instance, the antibody (i) shows decreased binding toGITR-positive, OX40-negative cells as compared to a monospecificbivalent antibody that binds to human GITR and contains the same VH andVL as the second antigen-binding domain that binds to human GITR; and/or(ii) shows decreased binding to GITR-negative, OX40-positive cells ascompared to a monospecific bivalent antibody that binds to human OX40and contains the same VH and VL as the first antigen-binding domain thatbinds to human OX40.

In one instance, the antibody (i) induces stronger natural killercell-mediated cytotoxicity towards regulatory T cells as compared to amonospecific bivalent antibody that binds to human GITR and contains thesame VH and VL as the second antigen-binding domain that binds to humanGITR; and/or (ii) induces stronger natural killer cell-mediatedcytotoxicity towards regulatory T cells as compared to a monospecificbivalent antibody that binds to human OX40 and contains the same VH andVL as the first antigen-binding domain that binds to human OX40.

In one instance, the antibody inhibits binding of human GITR ligand tohuman GITR. In one instance, the antibody inhibits binding of human OX40ligand to human OX40.

In one instance, the antibody, when bound to activated regulatory Tcells, binds to activating Fc gamma receptors selected from the groupconsisting of CD16, CD32A and CD64 to a greater extent than theantibody, when bound to activated effector T cells, binds to theactivating Fc gamma receptors selected from the group consisting ofCD16, CD32A and CD64. In one instance, the activating Fc gamma receptoris expressed on a cell selected from the group consisting ofmyeloid-derived effector cells and lymphocyte-derived effector cells.

In one instance, the antibody is agonistic to human GITR and/or humanOX40. In one instance, the antibody induces, activates, or enhances anactivity of human GITR. In one instance, the antibody induces,activates, or enhances an activity of human OX40.

In one instance, the first antigen-binding domain comprises a human IgG₁heavy chain constant region that comprises a N297A mutation or a N297Qmutation and/or the second antigen-binding domain comprises a human IgG₁heavy chain constant region that comprises a N297A mutation or a N297Qmutation, numbered according to the EU numbering system.

In one instance, the antibody is antagonistic to human GITR and/or humanOX40. In one instance, the antibody deactivates, reduces, or inhibits anactivity of human GITR. In one instance, the antibody deactivates,reduces, or inhibits an activity of human OX40. In one instance, theantibody inhibits or reduces human GITR signaling. In one instance, theantibody inhibits or reduces human OX40 signaling. In one instance, theantibody inhibits or reduces human GITR signaling induced by human GITRligand. In one instance, the antibody inhibits or reduces human OX40signaling induced by human OX40 ligand.

In one instance, the antibody decreases CD4+ T cell proliferationinduced by synovial fluid from rheumatoid arthritis patients. In oneinstance, the antibody increases survival of NOG mice transplanted withhuman PBMCs. In one instance, the antibody increases proliferation ofregulatory T cells in a GVHD model.

In one instance, the antibody further comprises a detectable label.

Also provided herein are compositions. In one instance, the compositioncomprises (i) a nucleic acid molecule encoding the light chain variableregion or light chain of the first antigen-binding fragment of anantibody provided herein, (ii) a nucleic acid molecule encoding theheavy chain variable region or heavy chain of the first antigen-bindingfragment of an antibody provided herein, (iii) a nucleic acid moleculeencoding the light chain variable region or light chain of the secondantigen-binding fragment of an antibody provided herein, and (iv) anucleic acid molecule encoding the heavy chain variable region or heavychain of the second antigen-binding fragment of an antibody providedherein.

Also provided herein are host cells. In one instance, a host cellcomprises (i) a nucleic acid molecule encoding the light chain variableregion or light chain of the first antigen-binding fragment of anantibody provided herein, (ii) a nucleic acid molecule encoding theheavy chain variable region or heavy chain of the first antigen-bindingfragment of an antibody provided herein, (iii) a nucleic acid moleculeencoding the light chain variable region or light chain of the secondantigen-binding fragment of an antibody provided herein, and (iv) anucleic acid molecule encoding the heavy chain variable region or heavychain of the second antigen-binding fragment of an antibody providedherein. In one instance, the host cell is selected from the groupconsisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO,YB/20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2/0, R1.1,B-W, L-M, COS 1, COS 7, BSC1, BSC40, BMT10 cell, plant cell, insectcell, and human cell in tissue culture. Also provided herein are methodsof making the multispecific (e.g., bispecific) antibodies that bind toGITR and/or OX40. In one instance, the method comprises culturing a hostcell provided herein so that the nucleic acid molecules are expressedand the antibody is produced

Also provided herein are methods of using the multispecific (e.g.,bispecific) antibodies that bind to GITR and/or OX40. In one instance, amethod for detecting cells expressing GITR and OX40 in a samplecomprises contacting the sample with an antibody provided herein.

Also provided herein are pharmaceutical compositions. In one instance, apharmaceutical composition comprises an antibody provided herein and apharmaceutically acceptable excipient.

Also provided herein are kits. In one instance, a kit comprises anantibody or pharmaceutical composition provided herein and a) adetection reagent, b) a GITR and/or OX40 antigen, c) a notice thatreflects approval for use or sale for human administration, or d) acombination thereof.

Also provided herein are methods of using the antibodies andpharmaceutical compositions provided herein. In one instance, a methodof modulating an immune response in a subject comprises administering tothe subject an effective amount of an antibody or pharmaceuticalcomposition provided herein. In one instance, a method for enhancing orinducing an immune response in a subject comprises administering to thesubject an effective amount of an antibody or pharmaceutical compositionprovided herein.

In one instance, a method of treating cancer in a subject comprisesadministering to the subject an effective amount of an antibody orpharmaceutical composition provided herein. In one instance, the canceris selected from the group consisting of melanoma, renal cancer,prostate cancer, colon cancer, and lung cancer. In one instance, themethod further comprises administering to the subject an inhibitor ofindoleamine-2,3-dioxygenase (IDO). In one instance, the inhibitor isepacadostat. In one instance, the inhibitor is F001287. In one instance,the inhibitor is indoximod. In one instance, the inhibitor is NLG919. Inone instance, the method further comprises administering to the subjecta vaccine. In one instance, the vaccine comprises a heat shock proteinpeptide complex (HSPPC) comprising a heat shock protein complexed withan antigenic peptide. In one instance, the heat shock protein is hsp70or hsc70 and is complexed with a tumor-associated antigenic peptide. Inone instance, the heat shock protein is gp96 protein and is complexedwith a tumor-associated antigenic peptide, wherein the HSPPC is derivedfrom a tumor obtained from a subject. In one instance, the methodfurther comprises administering to the subject a checkpoint targetingagent. In one instance, the checkpoint targeting agent is selected fromthe group consisting of an antagonist anti-PD-1 antibody, an antagonistanti-PD-L1 antibody, an antagonist anti-PD-L2 antibody, an antagonistanti-CTLA-4 antibody, an antagonist anti-TIM-3 antibody, an antagonistanti-LAG-3 antibody, an antagonist anti-CEACAM1 antibody, an agonistanti-GITR antibody, and an agonist anti-OX40 antibody.

In one instance, a method of treating an infectious disease comprisesadministering to the subject an effective amount of an antibody orpharmaceutical composition provided herein.

In one instance, a method for reducing or inhibiting an immune responsein a subject comprises administering to the subject an effective amountof an antibody or pharmaceutical composition provided herein.

In one instance, a method for treating an autoimmune or inflammatorydisease or disorder in a subject comprises administering to the subjectan effective amount of an antibody or pharmaceutical compositionprovided herein. In one instance, the autoimmune or inflammatory diseaseor disorder is selected from the group consisting of transplantrejection, graft-versus-host disease, vasculitis, asthma, rheumatoidarthritis, dermatitis, inflammatory bowel disease, uveitis, lupus,colitis, diabetes, multiple sclerosis, and airway inflammation.

In one instance, the subject is human.

6. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B: FIG. 1A is a set of histograms showing the expressionof GITR and OX40 on intratumoral effector T cells (Teff: CD4+ CD127+CD25+/− FOXP3−) and regulatory T cells (Treg: CD4+ CD127− CD25+ FOXP3+)from endometrial cancer tumor tissue, renal cell carcinoma (RCC) tumortissue, and non-small cell lung cancer (NSCLC) tumor tissue. FIG. 1B isa set of bar graphs showing the predicted number of GITR and OX40receptors on the surface of Tregs and Teff cells from ovarian cancertumor tissue, colorectal cancer (CRC) tumor tissue, endometrial cancertumor tissue, RCC tumor tissue, and NSCLC tumor tissue.

FIGS. 2A, 2B, and 2C are graphs showing the binding of test antibodiesto activated Hut102 cells that co-expressed GITR and OX40 (FIG. 2A),Jurkat cells expressing GITR (FIG. 2B), and Jurkat cells expressing OX40(FIG. 2C). The mean fluorescence intensity (MFI) is plotted against arange of antibody concentrations. The test antibodies used were DuoBodypab1876×pab2049 (FIGS. 2A-2C), a bivalent monospecific antibody pab1876(FIGS. 2A and 2B), a bivalent monospecific antibody pab2049 (FIGS. 2Aand 2C), DuoBody pab1876×isotype (FIG. 2A), DuoBody pab2049×isotype(FIG. 2A), and an isotype control antibody (FIGS. 2A-2C).

FIG. 3A is a set of histograms showing the expression of GITR and OX40on activated natural regulatory T (nTreg) cells. FIG. 3B is the resultof a reporter assay where test antibodies were examined for theirability to activate a reporter cell line expressing FcγRIIIA^(V158) whenthe antibodies were bound to activated nTregs. The test antibodies usedwere DuoBody pab1876×pab2049, the bivalent monospecific antibodypab1876, the bivalent monospecific antibody pab2049, and an isotypecontrol antibody. The relative light units (RLU) are normalized to theRLU values in the samples treated with the isotype control antibody atthe highest concentration tested and plotted against a dose titration ofantibody concentrations. FIGS. 3C and 3D are the result from a similarreporter assay where test antibodies were examined for their ability toactivate FcγRIIIA^(V158)-expressing reporter cells when the antibodieswere bound to Jurkat cells expressing GITR (FIG. 3C) or Jurkat cellsexpressing OX40 (FIG. 3D). The test antibodies used were DuoBodypab1876×pab2049, the bivalent monospecific antibody pab1876(F405L/F405L), the bivalent monospecific antibody pab2049 (K409R/K409R),and an isotype control antibody. RLUs are plotted against a range ofantibody concentrations. FIGS. 3E, 3F, and 3G are the result from anassay measuring NK cell-mediated lysis of activated effector T cells oractivated Tregs in the presence of an isotype control antibody, pab1876,pab2049, DuoBody pab1876×pab2049, or a combination of pab1876 andpab2049. FIG. 3E is a pair of histograms showing the expression of GITR(left) or OX40 (right) on activated effector T cells or Tregs asmeasured by flow cytometry. In FIGS. 3F and 3G, % cytotoxicity isplotted against a titration of antibody concentrations.

FIGS. 4A and 4B are graphs depicting the functional activity of DuoBodypab1876×pab2049 on primary human T cells from two donors followingStaphylococcus Enterotoxin A (SEA) stimulation. The concentration ofIL-2 is plotted at a dose titration of DuoBody pab1876×pab2049, DuoBodypab2049×isotype and an isotype control antibody.

FIGS. 5A and 5B: FIG. 5A is the result of a reporter assay where DuoBodypab1876×pab2049, trimeric GITRL, and an isotype control antibody weretested for their ability to activate Jurkat-huGITR-NF-κB-luciferasereporter cells. The relative light units (RLU), are plotted against arange of antibody or GITRL concentrations. FIG. 5B is the result of areporter assay where DuoBody pab1876×pab2049 and an isotype controlantibody were examined for their capacity to block GITRL-induced NF-κBsignaling. In this report assay, Jurkat-huGITR-NF-κB-luciferase reportercells were pre-incubated with DuoBody pab1876×pab2049 or an isotypecontrol antibody before activated by trimeric GITRL. The % GITRLactivity in the presence of a dose titration of antibody concentrationsis shown.

FIGS. 6A and 6B: FIG. 6A depicts NF-κB-luciferase signal fromJurkat-huOX40-NF-κB-luciferase reporter cells triggered by multimericOX40L, DuoBody pab1876×pab2049 or an isotype control antibody. RLUs areplotted against a dose titration of OX40L or antibody concentrations.FIG. 6B is the result of a reporter assay whereJurkat-huOX40-NF-κB-luciferase reporter cells were pre-incubated withDuoBody pab1876×pab2049 or an isotype control antibody before activatedby multimeric OX40L. The % OX40L activity is plotted against a range ofantibody concentrations.

FIG. 7 is a histogram showing the loss of binding of 1624-5 pre-B cellsexpressing the chimeric parental 231-32-15 antibody to biotinylated GITR(GITR-bio) when GITR-bio was pre-incubated with chimeric parental231-32-15, pab1875 or pab1876 antibodies. FIG. 7 right-hand profiledepicts the binding of 1624-5 pre-B cells expressing the chimericparental 231-32-15 antibody to GITR-bio. In the left-hand profile,however, there is loss of binding of 1624-5 cells expressing thechimeric parental 231-32-15 antibody to GITR-bio followingpre-incubation of GITR-bio with either the chimeric parental 231-32-15,pab1875 or pab1876 antibodies.

FIG. 8 shows the results of an epitope competition assay measured bysurface plasmon resonance (BIAcore® T100/200). GITR antigen wasimmobilized on a CM5 sensor chip and the anti-GITR antibodies applied ata concentration of 300 nM. Chimeric parental 231-32-15 antibody wasapplied first followed by the application of the murine antibody 6C8.

FIGS. 9A and 9B are the results of an epitope mapping experiment using acellular library expressing GITR variants generated by error prone PCR.Shown in FIGS. 9A and 9B is an alignment of sequences from the GITRvariants that bind to a polyclonal anti-GITR antibody but do not bind tothe anti-GITR chimeric parental 231-32-15 antibody.

FIGS. 10A and 10B are the result of an epitope mapping experiment usingalanine scanning. The following positions in human GITR (numberedaccording to SEQ ID NO:41) were separately mutated to an Alanine: P28A,T29A, G30A, G31A, P32A, T54A, T55A, R56A, C57A, C58A, R59A, D60A, Y61A,P62A, G63A, E64A, E65A, C66A, C67A, S68A, E69A, W70A, D71A, C72A, M73A,C74A, V75A and Q76A. The antibodies tested in the experiment shown inFIG. 10A included: the monoclonal anti-GITR antibodies pab1876, pab1967,pab1975, pab1979 and m6C8; and a polyclonal anti-GITR antibody (AF689,R&D systems). FIG. 10A is a table summarizing the binding of pab1876,pab1967, pab1975, pab1979 and the reference antibody m6C8 to 1624-5cells expressing human GITR alanine mutants. FIG. 10B is a set of flowcytometry plots showing the staining of 1624-5 cells expressing wildtype human GITR, D60A mutant, or G63A mutant using the monoclonalantibody 231-32-15, pab1876, or m6C8, or a polyclonal antibody. Thepercentage of GITR positive cells is indicated in each plot.

FIG. 11A is a sequence alignment of human GITR, VIM cynomolgus GITR, andV1M/Q62P/S63G cynomolgus GITR, highlighting the positions 62 and 63where two amino acids from cynomolgus GITR (GlnSer) were replaced bycorresponding residues in human GITR (ProGly). FIG. 11B is a set of flowcytometry plots showing the staining of 1624-5 cells expressing humanGITR, VIM cynomolgus GITR, or V1M/Q62P/S63G cynomolgus GITR using themonoclonal antibody 231-32-15, pab1876, or m6C8, or a polyclonalanti-GITR antibody.

FIG. 12 is a table summarizing the binding of the monoclonal anti-OX40antibodies pab1949w, pab2049, and pab1928 to 1624-5 cells expressinghuman OX40 alanine mutants.

7. DETAILED DESCRIPTION

Provided herein are multispecific antibodies (e.g., bispecificantibodies) that specifically bind to GITR (e.g., human GITR) and/orOX40 (e.g., human OX40).

For example, multispecific (e.g., bispecific) antibodies provided hereincan contain a first antigen-binding domain that binds to OX40 and asecond antigen-binding domain. The second antigen-binding domain can bedistinct from the first antigen-binding domain. The secondantigen-binding domain can bind to a different antigen (i.e., an antigenthat is not OX40) than the first antigen-binding domain. The secondantigen-binding domain can bind to a different epitope than the firstantigen-binding domain. In one instance, antibodies provided hereincontain a first antigen-binding domain that specifically binds to OX40(e.g., human OX40) and a second antigen-binding domain that specificallybinds to a TNFR superfamily protein. The TNFR superfamily protein canbe, for example, GITR, OX40, CD137, DR3, CD40, BAFFR, CD27, or HVEM. Inone instance, antibodies provided herein contain a first antigen-bindingdomain that specifically binds to OX40 (e.g., human OX40) and a secondantigen-binding domain that specifically binds to GITR (e.g., humanGITR).

In another example, antibodies provided herein can contain a firstantigen-binding domain and a second antigen-binding domain that binds toGITR. The first antigen-binding domain can be distinct from the secondantigen-binding domain. The first antigen-binding domain can bind to adifferent antigen (i.e., an antigen that is not GITR) than the firstantigen-binding domain. The second antigen-binding domain can bind to adifferent epitope than the first antigen-binding domain. In oneinstance, antibodies provided herein contain a first antigen-bindingdomain that specifically binds to a TNFR superfamily protein and asecond antigen-binding domain that specifically binds to GITR (e.g.,human GITR). The TNFR superfamily protein can be, for example, GITR,OX40, CD137, DR3, CD40, BAFFR, CD27, or HVEM. In another example,antibodies provided herein contain a first antigen-binding domain thatbinds to OX40 and a second antigen-binding domain that binds to GITR. Inone instance, antibodies provided herein contain a first antigen-bindingdomain that specifically binds to OX40 (e.g., human OX40) and a secondantigen-binding domain that specifically binds to GITR (e.g., humanGITR).

Also provided herein are antibodies that comprise an antigen-bindingdomain that specifically binds to OX40 (e.g., human OX40) and a TNFsuperfamily protein. Such antibodies can bind to cells expressing OX40(e.g., human OX40) and a receptor for the TNF superfamily protein. Alsoprovided herein are antibodies that comprise a TNF superfamily proteinand an antigen-binding domain that specifically binds to GITR (e.g.,human GITR). Such antibodies can bind to cells expressing a receptor forthe TNF superfamily protein and GITR (e.g., human GITR). The TNFsuperfamily protein can be, for example, GITR ligand, OX40 ligand, CD137ligand, DR3 ligand, CD40 ligand, BAFFR ligand, CD27 ligand, or HVEMligand. A TNF superfamily protein can replace the first antigen-bindingdomain or the second antigen-binding domain in any multispecific (e.g.,bispecific) antibody provided herein.

In one aspect, provided herein is a multispecific (e.g., bispecific)antibody that specifically binds to GITR and OX40 and enhances, induces,or increases one or more GITR and/or OX40 activities. In another aspect,provided herein is a multispecific (e.g., bispecific) antibody thatspecifically binds to GITR and OX40 and reduces, inhibits, or decreasesone or more GITR or OX40 activities. In a specific embodiment, theantibody is isolated.

Also provided are isolated nucleic acids (polynucleotides), such ascomplementary DNA (cDNA), encoding such antibodies. Further provided arevectors (e.g., expression vectors) and cells (e.g., host cells)comprising nucleic acids (polynucleotides) encoding such antibodies.Also provided are methods of making such antibodies. In other aspects,provided herein are methods and uses for inducing, increasing, orenhancing GITR and/or OX40 activity, and treating certain conditions,such as cancer. Further provided are methods and uses for inhibiting,decreasing, or reducing GITR and/or OX40 activity, and treating certainconditions, such as inflammatory or autoimmune diseases and disorders.Related compositions (e.g., pharmaceutical compositions), kits, anddetection methods are also provided.

7.1 Terminology

As used herein, the terms “about” and “approximately,” when used tomodify a numeric value or numeric range, indicate that deviations of 5%to 10% above and 5% to 10% below the value or range remain within theintended meaning of the recited value or range.

As used herein, B is a “substantially increasing function” of A over aspecified domain of A values if B substantially increases as A increasesover the specified domain, e.g., in a given experiment, or using meanvalues from multiple experiments. This definition allows for a value ofB corresponding to a specified value of A to be up to 1%, 2%, 3%, 4%,5%, 10%, 15%, or 20% lower relative to a value of B corresponding to anylower value of A.

As used herein, B is a “substantially decreasing function” of A over aspecified domain of A values if B substantially decreases as A increasesover the specified domain, e.g., in a given experiment, or using meanvalues from multiple experiments. This definition allows for a value ofB corresponding to a specified value of A to be up to 1%, 2%, 3%, 4%,5%, 10%, 15%, or 20% higher relative to a value of B corresponding toany lower value of A.

As used herein, the terms “antibody” and “antibodies” are terms of artand can be used interchangeably herein and refer to a molecule with anantigen-binding site that specifically binds an antigen.

Antibodies can include, for example, monoclonal antibodies,recombinantly produced antibodies, human antibodies, humanizedantibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins,synthetic antibodies, tetrameric antibodies comprising two heavy chainand two light chain molecules, an antibody light chain monomer, anantibody heavy chain monomer, an antibody light chain dimer, an antibodyheavy chain dimer, an antibody light chain-antibody heavy chain pair,intrabodies, heteroconjugate antibodies, single domain antibodies,monovalent antibodies, single chain antibodies or single-chain Fvs(scFv), camelized antibodies, affybodies, Fab fragments, F(ab′)₂fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id)antibodies (including, e.g., anti-anti-Id antibodies), bispecificantibodies, and multi-specific antibodies. In certain embodiments,antibodies described herein refer to polyclonal antibody populations.Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY),any class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, or IgA₂), or any subclass(e.g., IgG_(2a) or IgG_(2b)) of immunoglobulin molecule. In certainembodiments, antibodies described herein are IgG antibodies, or a class(e.g., human IgG₁, IgG₂, or IgG₄) or subclass thereof. In a specificembodiment, the antibody is a humanized monoclonal antibody. In anotherspecific embodiment, the antibody is a human monoclonal antibody, e.g.,that is an immunoglobulin. In certain embodiments, an antibody describedherein is an IgG₁, IgG₂, or IgG₄ antibody.

“Multispecific” antibodies are antibodies with at least two differentantigen-binding sites. Multispecific antibodies include bispecificantibodies that contain two different antigen-binding sites (exclusiveof the Fc region). Multispecific antibodies can include, for example,recombinantly produced antibodies, human antibodies, humanizedantibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins,synthetic antibodies, tetrameric antibodies comprising two heavy chainand two light chain molecules, an antibody light chain monomer,heteroconjugate antibodies, linked single chain antibodies orlinked-single-chain Fvs (scFv), camelized antibodies, affybodies, linkedFab fragments, F(ab′)₂ fragments, chemically-linked Fvs, anddisulfide-linked Fvs (sdFv). Multispecific antibodies can be of any type(e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, or IgA₂), or any subclass (e.g., IgG_(2a) or IgG_(2b))of immunoglobulin molecule. In certain embodiments, multispecificantibodies described herein are IgG antibodies, or a class (e.g., humanIgG₁, IgG₂, or IgG₄) or subclass thereof.

As used herein, the terms “antigen-binding domain,” “antigen-bindingregion,” “antigen-binding site,” and similar terms refer to the portionof antibody molecules which comprises the amino acid residues thatconfer on the antibody molecule its specificity for the antigen (e.g.,the complementarity determining regions (CDR)). The antigen-bindingregion can be derived from any animal species, such as rodents (e.g.,mouse, rat, or hamster) and humans.

A used herein, the term “anti-GITR/OX40” antibody refers to amultispecific antibody (e.g., a bispecific antibody) that contains anantigen-binding domain that binds to GITR (e.g., human GITR) and anantigen-binding domain that binds to OX40 (e.g., human OX40).

As used herein, the terms “variable region” or “variable domain” areused interchangeably and are common in the art. The variable regiontypically refers to a portion of an antibody, generally, a portion of alight or heavy chain, typically about the amino-terminal 110 to 125amino acids in the mature heavy chain and about 90 to 115 amino acids inthe mature light chain, which differ extensively in sequence amongantibodies and are used in the binding and specificity of a particularantibody for its particular antigen. The variability in sequence isconcentrated in those regions called complementarity determining regions(CDRs) while the more highly conserved regions in the variable domainare called framework regions (FR). Without wishing to be bound by anyparticular mechanism or theory, it is believed that the CDRs of thelight and heavy chains are primarily responsible for the interaction andspecificity of the antibody with antigen. In certain embodiments, thevariable region is a human variable region. In certain embodiments, thevariable region comprises rodent or murine CDRs and human frameworkregions (FRs). In particular embodiments, the variable region is aprimate (e.g., non-human primate) variable region. In certainembodiments, the variable region comprises rodent or murine CDRs andprimate (e.g., non-human primate) framework regions (FRs).

The terms “VL” and “VL domain” are used interchangeably to refer to thelight chain variable region of an antibody.

The terms “VH” and “VH domain” are used interchangeably to refer to theheavy chain variable region of an antibody.

The term “Kabat numbering” and like terms are recognized in the art andrefer to a system of numbering amino acid residues in the heavy andlight chain variable regions of an antibody, or an antigen-bindingportion thereof. In certain aspects, the CDRs of an antibody can bedetermined according to the Kabat numbering system (see, e.g., Kabat E A& Wu T T (1971) Ann NY Acad Sci 190: 382-391 and Kabat E A et al.,(1991) Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242). Using the Kabat numbering system, CDRs within an antibodyheavy chain molecule are typically present at amino acid positions 31 to35, which optionally can include one or two additional amino acids,following 35 (referred to in the Kabat numbering scheme as 35A and 35B)(CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions95 to 102 (CDR3). Using the Kabat numbering system, CDRs within anantibody light chain molecule are typically present at amino acidpositions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), andamino acid positions 89 to 97 (CDR3). In a specific embodiment, the CDRsof the antibodies described herein have been determined according to theKabat numbering scheme.

As used herein, the term “constant region” or “constant domain” areinterchangeable and have its meaning common in the art. The constantregion is an antibody portion, e.g., a carboxyl terminal portion of alight and/or heavy chain which is not directly involved in binding of anantibody to antigen but which can exhibit various effector functions,such as interaction with the Fc receptor. The constant region of animmunoglobulin molecule generally has a more conserved amino acidsequence relative to an immunoglobulin variable domain.

As used herein, the term “heavy chain” when used in reference to anantibody can refer to any distinct type, e.g., alpha (α), delta (δ),epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence ofthe constant domain, which give rise to IgA, IgD, IgE, IgG, and IgMclasses of antibodies, respectively, including subclasses of IgG, e.g.,IgG₁, IgG₂, IgG₃, and IgG₄.

As used herein, the term “light chain” when used in reference to anantibody can refer to any distinct type, e.g., kappa (κ) or lambda (λ)based on the amino acid sequence of the constant domains. Light chainamino acid sequences are well known in the art. In specific embodiments,the light chain is a human light chain.

As used herein, the term “EU numbering system” refers to the EUnumbering convention for the constant regions of an antibody, asdescribed in Edelman, G. M. et al., Proc. Natl. Acad. USA, 63, 78-85(1969) and Kabat et al, Sequences of Proteins of Immunological Interest,U.S. Dept. Health and Human Services, 5th edition, 1991, each of whichis herein incorporated by reference in its entirety.

“Binding affinity” generally refers to the strength of the sum total ofnon-covalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (K_(D)). Affinity can be measured and/or expressedin a number of ways known in the art, including, but not limited to,equilibrium dissociation constant (K_(D)), and equilibrium associationconstant (K_(A)). The K_(D) is calculated from the quotient ofk_(off)/k_(on), whereas K_(A) is calculated from the quotient ofk_(on)/k_(off). k_(on) refers to the association rate constant of, e.g.,an antibody to an antigen, and k_(off) refers to the dissociation of,e.g., an antibody to an antigen. The k_(on), and k_(off) can bedetermined by techniques known to one of ordinary skill in the art, suchas BIAcore® or KinExA.

As used herein, a “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having side chainshave been defined in the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Incertain embodiments, one or more amino acid residues within a CDR(s) orwithin a framework region(s) of an antibody can be replaced with anamino acid residue with a similar side chain.

As used herein, an “epitope” is a term in the art and refers to alocalized region of an antigen to which an antibody can specificallybind. An epitope can be, for example, contiguous amino acids of apolypeptide (linear or contiguous epitope) or an epitope can, forexample, come together from two or more non-contiguous regions of apolypeptide or polypeptides (conformational, non-linear, discontinuous,or non-contiguous epitope). In certain embodiments, the epitope to whichan antibody binds can be determined by, e.g., NMR spectroscopy, X-raydiffraction crystallography studies, ELISA assays, hydrogen/deuteriumexchange coupled with mass spectrometry (e.g., liquid chromatographyelectrospray mass spectrometry), array-based oligo-peptide scanningassays, and/or mutagenesis mapping (e.g., site-directed mutagenesismapping). For X-ray crystallography, crystallization may be accomplishedusing any of the known methods in the art (e.g., Giegé R et al., (1994)Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A(1990) Eur J Biochem 189: 1-23; Chayen N E (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303).Antibody:antigen crystals can be studied using well known X-raydiffraction techniques and can be refined using computer software suchas X-PLOR (Yale University, 1992, distributed by Molecular Simulations,Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff H Wet al.,; U.S. 2004/0014194), and BUSTER (Bricogne G (1993) ActaCrystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) MethEnzymol 276A: 361-423, ed Carter C W; Roversi P et al., (2000) ActaCrystallogr D Biol Crystallogr 56(Pt 10): 1316-1323). Mutagenesismapping studies can be accomplished using any method known to one ofskill in the art. See, e.g., Champe M et al., (1995) J Biol Chem 270:1388-1394 and Cunningham B C & Wells J A (1989) Science 244: 1081-1085for a description of mutagenesis techniques, including alanine scanningmutagenesis techniques. In a specific embodiment, the epitope of anantibody is determined using alanine scanning mutagenesis studies.

As used herein, the terms “immunospecifically binds,”“immunospecifically recognizes,” “specifically binds,” and “specificallyrecognizes” are analogous terms in the context of antibodies and referto molecules that bind to an antigen (e.g., epitope or immune complex)as such binding is understood by one skilled in the art. For example, amolecule that specifically binds to an antigen can bind to otherpeptides or polypeptides, generally with lower affinity as determinedby, e.g., immunoassays, BIAcore®, KinExA 3000 instrument (SapidyneInstruments, Boise, ID), or other assays known in the art. In a specificembodiment, molecules that immunospecifically bind to an antigen bind tothe antigen with a K_(A) that is at least 2 logs, 2.5 logs, 3 logs, 4logs or greater than the K_(A) when the molecules bind non-specificallyto another antigen. In the context of multispecific (e.g., bispecific)antibodies, the terms “immunospecifically binds,” “immunospecificallyrecognizes,” “specifically binds,” and “specifically recognizes” referto antibodies that have distinct specificities for more than one antigenor for more than one epitope on a single antigen. For example, abispecific antibody may, e.g., specifically bind each of human OX40 andhuman GITR, e.g., with distinct antigen-binding domains.

In another specific embodiment, antigen-binding domains thatimmunospecifically bind to an antigen do not cross react with otherproteins under similar binding conditions. In another specificembodiment, antigen-binding domains that immunospecifically bind to GITRantigen do not cross react with other non-GITR proteins. In anotherspecific embodiment, antigen-binding domains that immunospecificallybind to OX40 antigen do not cross react with other non-OX40 proteins. Ina specific embodiment, provided herein is an antibody containing anantigen-binding domain that binds to GITR or OX40 with higher affinitythan to another unrelated antigen. In certain embodiments, providedherein is an antibody containing an antigen-binding domain that binds toGITR or OX40 (e.g., human GITR or human OX40) with a 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higheraffinity than to another, unrelated antigen as measured by, e.g., aradioimmunoassay, surface plasmon resonance, or kinetic exclusion assay.In a specific embodiment, the extent of binding of an anti-GITRantigen-binding domain described herein to an unrelated, non-GITRprotein is less than 10%, 15%, or 20% of the binding of theantigen-binding domain to GITR protein as measured by, e.g., aradioimmunoassay. In a specific embodiment, the extent of binding of ananti-OX40 antigen-binding domain described herein to an unrelated,non-OX40 protein is less than 10%, 15%, or 20% of the binding of theantigen-binding domain to OX40 protein as measured by, e.g., aradioimmunoassay.

In a specific embodiment, provided herein is an antibody containing anantigen-binding domain that binds to human GITR with higher affinitythan to another species of GITR and/or an antigen-binding domain thatbinds to human OX40 with higher affinity than to another species ofOX40. In certain embodiments, provided herein is an antibody containingan antigen-binding domain that binds to human GITR with a 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or higher affinitythan to another species of GITR as measured by, e.g., aradioimmunoassay, surface plasmon resonance, or kinetic exclusion assayand/or that binds to human OX40 with a 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70% or higher affinity than to anotherspecies of OX40 as measured by, e.g., a radioimmunoassay, surfaceplasmon resonance, or kinetic exclusion assay. In a specific embodiment,an antibody described herein, which binds to human GITR and human OX40,will bind to another species of GITR and/or OX40 protein with less than10%, 15%, or 20% of the binding of the antibody to the human GITR and/orOX40 protein as measured by, e.g., a radioimmunoassay, surface plasmonresonance, or kinetic exclusion assay.

As used herein, the terms “glucocorticoid-induced TNFR family relatedreceptor” or “GITR” or “GITR polypeptide” refer to GITR including, butnot limited to, native GITR, an isoform of GITR, or an interspecies GITRhomolog of GITR. GITR is also known as activation-inducible TNFR familyreceptor (AITR), GITR-D, CD357, and tumor necrosis factor receptorsuperfamily member 18 (TNFRSF18). GenBank™ accession numbers B C152381and B C152386 provide human GITR nucleic acid sequences. Swiss-Protaccession number Q9Y5U5-1 (TNR18_HUMAN; SEQ ID NO:41) and GenBank™accession number NP_004186 provide exemplary human GITR amino acidsequences for isoform 1. This amino acid sequence is 241 amino acids inlength with the first 25 amino acid residues encoding the signalsequence. Isoform 1 is a type I membrane protein. An exemplary matureamino acid sequence of human GITR is provided as SEQ ID NO:40. Incontrast, isoform 2 is a secreted form of human GITR and isapproximately 255 amino acids in length. Swiss-Prot accession numberQ9Y5U5-2 and GenBank™ accession number NP_683699 provide exemplary humanGITR amino acid sequences for isoform 2. Isoform 3 of human GITR isapproximately 234 amino acids in length. Swiss-Prot accession numberQ9Y5U5-3 and GenBank™ accession number NP_683700 (isoform 3 precursor)provide exemplary human GITR amino acid sequences for isoform 3. In aspecific embodiment, the GITR is human GITR. In another specificembodiment, the GITR is human GITR isoform 1 (SEQ ID NO:41). In certainembodiments, the GITR is human isoform 2 (SEQ ID NO:42) or isoform 3(SEQ ID NO:43). Human GITR is designated GeneID: 8784 by Entrez Gene.SEQ ID NO:44 provides the cynomolgus GITR amino acid sequence, and aminoacids 26-234 of SEQ ID NO:44 represent the mature form of cynomolgusGITR. As used herein, the term “human GITR” refers to GITR comprisingthe polypeptide sequence of SEQ ID NO:40.

As used herein, the terms “GITR ligand” and “GITRL” refer toglucocorticoid-induced TNFR-related protein ligand. GITRL is otherwiseknown as activation-induced TNF-related ligand (AITRL) and tumornecrosis factor ligand superfamily member 18 (TNFSF18). GenBank™accession number AF125303 provides an exemplary human GITRL nucleic acidsequence. GenBank™ accession number NP_005083 and Swiss-Prot accessionnumber Q9UNG2 provide exemplary human GITRL amino acid sequences.

As used herein, the terms “OX40 receptor” or “OX40” or “OX40polypeptide” refer to OX40 including, but not limited to, native OX40,an isoform of OX40, or an interspecies OX40 homolog of OX40. OX40 isalso known as tumor necrosis factor receptor superfamily member 4(TNFRSF4), ACT35, CD134, IMD16, and TXGP1L. GenBank™ accession numbersBC105070 and BC105072 provide human OX40 nucleic acid sequences. Refseqnumber NP_003318.1 provides the amino acid sequence of human OX40. Theimmature amino acid sequence of human OX40 is provided as SEQ ID NO:73.The mature amino acid sequence of human OX40 is provided as SEQ IDNO:72. Human OX40 is designated GeneID: 7293 by Entrez Gene. RefSeqnumbers XM_005545122.1 and XP_005545179.1 provide predicted cynomolgusOX40 nucleic acid sequences and amino acid sequences, respectively. Asoluble isoform of human OX40 has also been reported (Taylor L et al.,(2001) J Immunol Methods 255: 67-72). As used herein, the term “humanOX40” refers to OX40 comprising the polypeptide sequence of SEQ IDNO:72.

As used herein, the terms “OX40 ligand” and “OX40L” refer to tumornecrosis factor ligand superfamily member 4 (TNFSF4). OX40L is otherwiseknown as CD252, GP34, TXGP1, and CD134L. GenBank™ accession numbersD90224.1 and AK297932.1 provide exemplary human OX40L nucleic acidsequences. RefSeq number NP_003317.1 and Swiss-Prot accession numberP23510-1 provide exemplary human OX40L amino acid sequences forisoform 1. RefSeq number NP_001284491.1 and Swiss-Prot accession numberP23510-2 provide exemplary human OX40L amino acid sequences for isoform2. Human OX40L is designated GeneID: 7292 by Entrez Gene.

As used herein, the term “host cell” can be any type of cell, e.g., aprimary cell, a cell in culture, or a cell from a cell line. In specificembodiments, the term “host cell” refers to a cell transfected with anucleic acid molecule and the progeny or potential progeny of such acell. Progeny of such a cell are not necessarily identical to the parentcell transfected with the nucleic acid molecule, e.g., due to mutationsor environmental influences that may occur in succeeding generations orintegration of the nucleic acid molecule into the host cell genome.

As used herein, the term “effective amount” in the context of theadministration of a therapy to a subject refers to the amount of atherapy that achieves a desired prophylactic or therapeutic effect.

As used herein, the terms “subject” and “patient” are usedinterchangeably. The subject can be an animal. In some embodiments, thesubject is a mammal such as a non-primate (e.g., cow, pig, horse, cat,dog, rat, etc.) or a primate (e.g., monkey or human), most preferably ahuman. In some embodiments, the subject is a cynomolgus monkey. Incertain embodiments, such terms refer to a non-human animal (e.g., anon-human animal such as a pig, horse, cow, cat, or dog). In someembodiments, such terms refer to a pet or farm animal. In specificembodiments, such terms refer to a human.

As used herein, the binding between a test antibody and a first antigenis “substantially weakened” relative to the binding between the testantibody and a second antigen if the binding between the test antibodyand the first antigen is reduced by at least 30%, 40%, 50%, 60%, 70%, or80% relative to the binding between the test antibody and the secondantigen, as measured in e.g., a flow cytometry analysis, or in a givenexperiment, or using mean values from multiple experiments, as assessedby, e.g., an assay comprising the following steps: (a) expressing on thesurface of cells (e.g., 1624-5 cells) the first antigen or the secondantigen; (b) staining the cells expressing the first antigen or thesecond antigen using, e.g., 2 μg/ml of the test antibody or a polyclonalantibody in a flow cytometry analysis and recording mean fluorescenceintensity (MFI) values, e.g., as the mean from more than onemeasurement, wherein the polyclonal antibody recognizes both the firstantigen and the second antigen; (c) dividing the MFI value of the testantibody for the cells expressing the second antigen by the MFI value ofthe polyclonal antibody for the cells expressing the second antigen (MFIratio₂); (d) dividing the MFI value of the test antibody for the cellsexpressing the first antigen by the MFI value of the polyclonal antibodyfor the cells expressing the first antigen (MFI ratio₁); and (e)determining the percentage of reduction in binding by calculating100%*(1−(MFI ratio₁/MFI ratio₂)).

The determination of “percent identity” between two sequences (e.g.,amino acid sequences or nucleic acid sequences) can also be accomplishedusing a mathematical algorithm. A specific, non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin S & Altschul S F (1990) PNAS 87: 2264-2268,modified as in Karlin S & Altschul S F (1993) PNAS 90: 5873-5877. Suchan algorithm is incorporated into the NBLAST and)(BLAST programs ofAltschul S F et al., (1990) J Mol Biol 215: 403. BLAST nucleotidesearches can be performed with the NBLAST nucleotide program parametersset, e.g., for score=100, wordlength=12 to obtain nucleotide sequenceshomologous to a nucleic acid molecules described herein. BLAST proteinsearches can be performed with the)(BLAST program parameters set, e.g.,to score 50, wordlength=3 to obtain amino acid sequences homologous to aprotein molecule described herein. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul S F et al., (1997) Nuc Acids Res 25: 3389 3402. Alternatively,PSI BLAST can be used to perform an iterated search which detectsdistant relationships between molecules (Id.). When utilizing BLAST,Gapped BLAST, and PSI Blast programs, the default parameters of therespective programs (e.g., of)(BLAST and NBLAST) can be used (see, e.g.,National Center for Biotechnology Information (NCBI) on the worldwideweb, ncbi.nlm.nih.gov). Another specific, non-limiting example of amathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, 1988, CABIOS 4:11 17. Such an algorithmis incorporated in the ALIGN program (version 2.0) which is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

7.2 Multispecific Antibodies that Bind to GITR and/or OX40

In a specific aspect, provided herein are multispecific antibodies(e.g., bispecific antibodies) which specifically bind to GITR and/orOX40 (e.g., human GITR and human OX40). For instance, a multispecific(e.g., bispecific) antibody provided herein can comprise a firstantigen-binding domain that binds to OX40 and a second antigen-bindingdomain. A multispecific (e.g., bispecific) antibody provided herein canalso comprise a first antigen-binding domain and a secondantigen-binding domain that binds to GITR. These multispecific (e.g.,bispecific) antibodies can also bind to other tumor necrosis factorreceptor (TNFR) superfamily proteins, e.g., those that are co-regulatedwith GITR and/or OX40. Such multispecific antibodies advantageously showgreater specificity for certain subsets of immune cells containing thecombination of target proteins than monospecific bivalent antibodiesthat only bind to one TNFR superfamily protein.

For example, provided herein are antibodies that comprise a firstantigen-binding domain that binds to OX40 and a second antigen-bindingdomain that binds to a tumor necrosis factor receptor (TNFR) superfamilyprotein, such as GITR, OX40, CD137, or DR3. In another example, providedherein are antibodies that comprise a first antigen-binding domain thatbinds to a TNFR superfamily protein, such as GITR, OX40, CD137, or DR3,and a second antigen-binding domain that binds to GITR.

Also provided herein are multispecific (e.g., bispecific) antibodiesthat comprise a first antigen-binding domain that binds to OX40 and asecond antigen-binding domain that binds to GITR.

The antibodies provided herein that contain an OX40 antigen-bindingdomain and a GITR antigen-binding domain can show increased binding tocells expressing GITR and OX40 (e.g., T regulatory cells) as compared,for example, to a monospecific bivalent antibody that binds to GITR andcontains the same GITR antigen-binding domain; and/or as compared to amonospecific bivalent antibody that binds to OX40 and contains the sameOX40 antigen-binding domain.

The antibodies provided herein that contain an OX40 antigen-bindingdomain and a GITR antigen-binding domain can also show decreased bindingto GITR-positive, OX40-negative cells (e.g., at low concentrations) ascompared to a monospecific bivalent antibody that binds to GITR andcontains the same GITR antigen-binding domain.

The antibodies provided herein that contain an OX40 antigen-bindingdomain and a GITR antigen-binding domain can also show decreased bindingto GITR-negative, OX40-positive cells (e.g., at low concentrations) ascompared to a monospecific bivalent antibody that binds to OX40 andcontains the same OX40 antigen-binding domain.

In certain embodiments, a multispecific (e.g., bispecific) antibodydescribed herein which specifically binds to GITR and OX40 can bind tohuman CD4+ T cells and human CD8+ T cells. In certain embodiments, anantibody described herein binds to human CD4+ cells and cynomolgusmonkey CD4+ T cells. The antibodies provided herein which specificallybind to GITR and OX40 can show enhanced binding to regulatory T cells ascompared to effector T cells. In some instances, the antibodies providedherein which specifically bind to GITR and OX40 show enhanced binding tointratumoral regulatory T cells as compared to intratumoral effector Tcells.

The multispecific (e.g., bispecific) antibodies provided herein thatspecifically bind to GITR and OX40 can inhibit binding of human GITRligand to human GITR and/or inhibit binding of human OX40 ligand tohuman OX40.

In one instance, an antibody provided herein that specifically binds toGITR and OX40 contains a combination of CDRs shown in a single row ofTable 1 below.

TABLE 1 CDR sequences of exemplary anti-GITR/OX40 antibodies *GITR-Binding Sequence SEQ ID NO. OX40-Binding Sequence SEQ ID NO. VH VHVH VL VL VL VH VH VH VL VL VL CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 CDR1 CDR2CDR3 CDR1 CDR2 CDR3 7 10 3 14 5 16 47 48 49 50 51 52 8 11 3 15 5 17 4748 49 50 51 52 9 12 3 14 5 16 47 48 49 50 51 52 9 13 3 14 5 16 47 48 4950 51 52 1 2 3 4 5 6 47 48 49 50 51 52 87 88 3 90 5 92 47 48 49 50 51 527 10 3 14 5 16 47 48 49 50 51 53 8 11 3 15 5 17 47 48 49 50 51 53 9 12 314 5 16 47 48 49 50 51 53 9 13 3 14 5 16 47 48 49 50 51 53 1 2 3 4 5 647 48 49 50 51 53 87 88 3 90 5 92 47 48 49 50 51 53 * The CDRs in Table1 are determined according to Kabat.

In one instance, an antibody provided herein that specifically binds toGITR and OX40 contains a combination of two heavy chain variable domainsand two light chain variable domains shown in a single row of Table 2below.

TABLE 2 Heavy chain variable domain (VH) and light chain variable domain(VL) sequences of exemplary anti-GITR/OX40 antibodies GITR VH GITR VLOX40 VH OX40 VL (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:) NO:) NO:) NO:) 1819 54 55 20 21 54 55 22 23 54 55 24 23 54 55 25 26 54 55 18 19 54 56 2021 54 56 22 23 54 56 24 23 54 56 25 26 54 56

In one instance, an antibody provided herein that specifically binds toGITR and OX40 contains a combination of two heavy chains and two lightchains shown in a single row of Table 3 below.

TABLE 3 Heavy chain (HC) and light chain (LC) sequences of exemplaryanti-GITR/OX40 DuoBody antibodies GITR HC GITR LC OX40 HC OX40 LC (SEQID (SEQ ID (SEQ ID (SEQ ID Antibody NO:) NO:) NO:) NO:) pab1876w (F405L)× pab2049w 31 37 61 67 (K409R) pab1876w (F405L/N297A) × 32 37 62 67pab2049w (K409R/N297A) pab1876w 33 37 63 67 (F405L/L234F/L235E/D265A) ×pab2049w (K409R/L234F/L235E/D265A) pab1876w (F405L) × pab1949w 31 37 6169 (K409R) pab1876w (F405L/N297A) × 32 37 62 69 pab1949w (K409R/N297A)pab1876w 33 37 63 69 (F405L/L234F/L235E/D265A) × pab1949w(K409R/L234F/L235E/D265A) pab1876w (K409R) × pab2049w 34 37 64 67(F405L) pab1876w (K409R/N297A) × 35 37 65 67 pab2049w (F405L/N297A)pab1876w 39 37 71 67 (K409R/L234F/L235E/D265A) × pab2049w(F405L/L234F/L235E/D265A) pab1876w (K409R) × pab1949w 34 37 64 69(F405L) pab1876w (K409R/N297A) × 35 37 65 69 pab1949w (F405L/N297A)pab1876w 39 37 71 69 (K409R/L234F/L235E/D265A) × pab1949w(F405L/L234F/L235E/D265A) pab1876w (F405L) × pab2049w 76 37 120 67(K409R) without heavy chain terminal lysine pab1876w (F405L/N297A) × 7737 121 67 pab2049w (K409R/N297A) without heavy chain terminal lysinepab1876w 78 37 122 67 (F405L/L234F/L235E/D265A) × pab2049w(K409R/L234F/L235E/D265A) without heavy chain terminal lysine pab1876w(F405L) × pab1949w 76 37 120 69 (K409R) without heavy chain terminallysine pab1876w (F405L/N297A) × 77 37 121 69 pab1949w (K409R/N297A)without heavy chain terminal lysine pab1876w 78 37 122 69(F405L/L234F/L235E/D265A) × pab1949w (K409R/L234F/L235E/D265A) withoutheavy chain terminal lysine pab1876w (K409R) × pab2049w 79 37 123 67(F405L) without heavy chain terminal lysine pab1876w (K409R/N297A) × 8037 124 67 pab2049w (F405L/N297A) without heavy chain terminal lysinepab1876w 82 37 83 67 (K409R/L234F/L235E/D265A) × pab2049w(F405L/L234F/L235E/D265A) without heavy chain terminal lysine pab1876w(K409R) × pab1949w 79 37 123 69 (F405L) without heavy chain terminallysine pab1876w (K409R/N297A) × 80 37 124 69 pab1949w (F405L/N297A)without heavy chain terminal lysine pab1876w 82 37 83 69(K409R/L234F/L235E/D265A) × pab1949w (F405L/L234F/L235E/D265A) withoutheavy chain terminal lysine

A multispecific antibody, e.g., a bispecific antibody, that binds toGITR and/or OX40 as provided herein can be prepared by chemicallylinking two different monoclonal antibodies or by fusing two hybridomacell lines to produce a hybrid-hybridoma. Other multivalent formats thatcan be used include, for example, Kλ-bodies, dAbs, diabodies, TandAbs,nanobodies, SMIPs, DNLs, strand-exchange engineered domain bodies(SEEDbodies), Affibodies, Fynomers, Kunitz Domains, Albu-dabs, DARTs,DVD-IG, Covx-bodies, peptibodies, scFv-Igs, SVD-Igs, dAb-Igs,Knobs-in-Holes, DuoBody antibodies and triomAbs. Exemplary bispecificformats are discussed in Garber et al., Nature Reviews Drug Discovery13:799-801 (2014), which is herein incorporated by reference in itsentirety.

Exemplary bispecific antibody molecules of the invention comprise (i) asingle antibody that has two arms comprising different antigen-bindingregions, one with a specificity to a first antigen such as OX40 and onewith a specificity to a second antigen such as GITR, (ii) a singleantibody that has one antigen-binding region or arm specific to a firstantigen such as OX40 and a second antigen-binding region or arm specificto a second antigen such as GITR, (iii) a single chain antibody that hasa first specificity to a first antigen such as OX40 and a secondspecificity to a second antigen such as GITR, e.g., via two scFvs linkedin tandem by an extra peptide linker; (iv) a dual-variable-domainantibody (DVD-Ig), where each light chain and heavy chain contains twovariable domains in tandem through a short peptide linkage (Wu et al.,Generation and Characterization of a Dual Variable Domain Immunoglobulin(DVD-Ig.™.) Molecule, In: Antibody Engineering, Springer BerlinHeidelberg (2010)); (v) a chemically-linked bispecific (Fab′)₂ fragment;(vi) a Tandab, which is a fusion of two single chain diabodies resultingin a tetravalent bispecific antibody that has two binding sites for eachof the target antigens; (vii) a flexibody, which is a combination ofscFvs with a diabody resulting in a multivalent molecule; (viii) a socalled “dock and lock” molecule, based on the “dimerization and dockingdomain” in Protein Kinase A, which, when applied to Fabs, can yield atrivalent bispecific binding protein consisting of two identical Fabfragments linked to a different Fab fragment; (ix) a so-called Scorpionmolecule, comprising, e.g., two scFvs fused to both termini of a humanFab-arm; and (x) a diabody.

Examples of different classes of bispecific antibodies include but arenot limited to IgG-like molecules with complementary CH3 domains toforce heterodimerisation; recombinant IgG-like dual targeting molecules,wherein the two sides of the molecule each contain the Fab fragment orpart of the Fab fragment of at least two different antibodies; IgGfusion molecules, wherein full length IgG antibodies are fused to extraFab fragment or parts of Fab fragment; Fc fusion molecules, whereinsingle chain Fv molecules or stabilized diabodies are fused toheavy-chain constant-domains, Fc-regions or parts thereof; Fab fusionmolecules, wherein different Fab-fragments are fused together; ScFv- anddiabody-based and heavy chain antibodies (e.g., domain antibodies,nanobodies) wherein different single chain Fv molecules or differentdiabodies or different heavy-chain antibodies (e.g. domain antibodies,nanobodies) are fused to each other or to another protein or carriermolecule.

Examples of Fab fusion bispecific antibodies include but are not limitedto F(ab)₂ (Medarex/AMGEN), Dual-Action or Bis-Fab (Genentech),Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) andFab-Fv (UCB-Celltech). Examples of ScFv-, diabody-based and domainantibodies include but are not limited to Bispecific T Cell Engager(BITE) (Micromet, Tandem Diabody (Tandab) (Affimed), Dual AffinityRetargeting Technology (DART) (MacroGenics), Single-chain Diabody(Academic), TCR-like Antibodies (AIT, ReceptorLogics), Human SerumAlbumin ScFv Fusion (Merrimack) and COMBODY (Epigen Biotech), dualtargeting nanobodies (Ablynx), and dual targeting heavy chain onlydomain antibodies.

In particular embodiments, a multispecific (e.g., bispecific) antibodycan be a chimeric antibody or a humanized antibody. In certainembodiments, a multispecific (e.g., bispecific) antibody can be aF(ab′)₂ fragment.

In certain embodiments, the multispecific (e.g., bispecific) antibody,that binds to GITR and/or OX40, is a DuoBody antibody.

In certain embodiments, a first antigen-binding domain that binds toOX40 as described herein comprises a human IgG₁ heavy chain constantregion comprising a F405L mutation, and a second antigen-binding domainthat binds to GITR as described herein comprises a human IgG₁ heavychain constant region comprising a K409R mutation, numbered according tothe EU numbering system.

In certain embodiments, a first antigen-binding domain that binds toOX40 as described herein comprises a human IgG₁ heavy chain constantregion comprising a K409R mutation, and a second antigen-binding domainthat binds to GITR as described herein comprises a human IgG₁ heavychain constant region comprising a F405L mutation, numbered according tothe EU numbering system.

As provided herein, multispecific antibodies (e.g. bispecificantibodies) that bind to GITR and/or OX40 can agonize or antagonize GITRand/or OX40 activity. Antibodies that agonize GITR and/or OX40 functioninclude antibodies that cluster GITR and/or OX40. Clustering can result,e.g., as a result of Fc-Fc receptor (FcR) interaction. Thus, antibodiesthat agonize GITR and/or OX40 include antibodies with increasedFc-receptor binding. Mutations that increase Fc-receptor binding areknown in the art and include, for example, antibodies with anafucosylated Fc, and antibodies with mutations such as S267E/L328F (theSELF mutant) and S239D/A330L/I332E, numbered according to the EUnumbering system. In some embodiments, a multispecific (e.g.,bispecific) agonist antibody that binds to GITR and/or OX40 comprises anIgG₂ constant region containing C127S, numbered according to Kabat.Antibodies that antagonize GITR and/or OX40 function include antibodieswith diminished Fc-receptor binding. Mutations that diminish Fc-receptorbinding are known in the art and include, for example, N297A; N297Q;D265A; L234F/L235E; L234F/L235E/N297Q; L234F/L235E/P331S; D265A/N297Q;and L234F/L235E/D265A/N297Q/P331S, numbered according to the EUnumbering system. In some embodiments, a multispecific (e.g.,bispecific) antagonist antibody that binds to GITR and/or OX40 comprisesan IgG1 constant region containing N297A, numbered according to the EUnumbering system. In some embodiments, a multispecific (e.g.,bispecific) antagonist antibody that binds to GITR and/or OX40 comprisesan IgG1 constant region containing N297Q, numbered according to the EUnumbering system. In some embodiments, a multispecific (e.g.,bispecific) antagonist antibody that binds to GITR and/or OX40 comprisesan IgG1 constant region containing D265A, numbered according to the EUnumbering system. In some embodiments, a multi specific (e.g.,bispecific) antagonist antibody that binds to GITR and/or OX40 comprisesan IgG1 constant region containing L234F/L235E/D265A, numbered accordingto the EU numbering system. In some embodiments, a multispecific (e.g.,bispecific) antagonist antibody that binds to GITR and/or OX40 comprisesan IgG1 constant region containing a mutation selected from the groupconsisting of D265A, P329A, and a combination thereof, numberedaccording to the EU numbering system.

In some embodiments, one, two, or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of an antibodydescribed herein (e.g., CH2 domain (residues 231-340 of human IgG₁)and/or CH3 domain (residues 341-447 of human IgG₁) and/or the hingeregion, with numbering according to the EU numbering system, e.g., toincrease or decrease the affinity of the antibody for an Fc receptor(e.g., an activated Fc receptor) on the surface of an effector cell.Mutations in the Fc region of an antibody that decrease or increase theaffinity of an antibody for an Fc receptor and techniques forintroducing such mutations into the Fc receptor or fragment thereof areknown to one of skill in the art. Examples of mutations in the Fcreceptor of an antibody that can be made to alter the affinity of theantibody for an Fc receptor are described in, e.g., Smith P et al.,(2012) PNAS 109: 6181-6186, U.S. Pat. No. 6,737,056, and InternationalPublication Nos. WO 02/060919; WO 98/23289; and WO 97/34631, which areincorporated herein by reference.

In a specific embodiment, one, two, or more amino acid mutations (i.e.,substitutions, insertions or deletions) are introduced into an IgGconstant domain, or FcRn-binding fragment thereof (preferably an Fc orhinge-Fc domain fragment) to alter (e.g., decrease or increase)half-life of the antibody in vivo. See, e.g., International PublicationNos. WO 02/060919; WO 98/23289; and WO 97/34631; and U.S. Pat. Nos.5,869,046, 6,121,022, 6,277,375 and 6,165,745 for examples of mutationsthat will alter (e.g., decrease or increase) the half-life of anantibody in vivo. In some embodiments, one, two or more amino acidmutations (i.e., substitutions, insertions, or deletions) are introducedinto an IgG constant domain, or FcRn-binding fragment thereof(preferably an Fc or hinge-Fc domain fragment) to decrease the half-lifeof the antibody in vivo. In other embodiments, one, two or more aminoacid mutations (i.e., substitutions, insertions or deletions) areintroduced into an IgG constant domain, or FcRn-binding fragment thereof(preferably an Fc or hinge-Fc domain fragment) to increase the half-lifeof the antibody in vivo. In a specific embodiment, the antibodies mayhave one or more amino acid mutations (e.g., substitutions) in thesecond constant (CH2) domain (residues 231-340 of human IgG₁) and/or thethird constant (CH3) domain (residues 341-447 of human IgG₁), withnumbering according to the EU numbering system. In a specificembodiment, the constant region of the IgG₁ of an antibody describedherein comprises a methionine (M) to tyrosine (Y) substitution inposition 252, a serine (S) to threonine (T) substitution in position254, and a threonine (T) to glutamic acid (E) substitution in position256, numbered according to the EU numbering system. See U.S. Pat. No.7,658,921, which is incorporated herein by reference. This type ofmutant IgG, referred to as “YTE mutant” has been shown to displayfourfold increased half-life as compared to wild-type versions of thesame antibody (see Dall'Acqua W F et al., (2006) J Biol Chem 281:23514-24). In certain embodiments, an antibody comprises an IgG constantdomain comprising one, two, three or more amino acid substitutions ofamino acid residues at positions 251-257, 285-290, 308-314, 385-389, and428-436, numbered according to the EU numbering system.

In a further embodiment, one, two, or more amino acid substitutions areintroduced into an IgG constant domain Fc region to alter the effectorfunction(s) of the antibody. For example, one or more amino acidsselected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and322, numbered according to the EU numbering system, can be replaced witha different amino acid residue such that the antibody has an alteredaffinity for an effector ligand but retains the antigen-binding abilityof the parent antibody. The effector ligand to which affinity is alteredcan be, for example, an Fc receptor or the C1 component of complement.This approach is described in further detail in U.S. Pat. Nos. 5,624,821and 5,648,260. In some embodiments, the deletion or inactivation(through point mutations or other means) of a constant region domain mayreduce Fc receptor binding of the circulating antibody therebyincreasing tumor localization. See, e.g., U.S. Pat. Nos. 5,585,097 and8,591,886 for a description of mutations that delete or inactivate theconstant domain and thereby increase tumor localization. In certainembodiments, one or more amino acid substitutions may be introduced intothe Fc region of an antibody described herein to remove potentialglycosylation sites on Fc region, which may reduce Fc receptor binding(see, e.g., Shields R L et al., (2001) J Biol Chem 276: 6591-604). Invarious embodiments, one or more of the following mutations in theconstant region of an antibody described herein may be made: an N297Asubstitution; an N297Q substitution; a L235A substitution and a L237Asubstitution; a L234A substitution and a L235A substitution; a E233Psubstitution; a L234V substitution; a L235A substitution; a C236deletion; a P238A substitution; a D265A substitution; a A327Qsubstitution; or a P329A substitution, numbered according to the EUnumbering system.

In a specific embodiment, an antibody described herein comprises theconstant domain of an IgG₁ with an N297Q or N297A amino acidsubstitution, numbered according to the EU numbering system.

In certain embodiments, one or more amino acids selected from amino acidresidues 329, 331, and 322 in the constant region of an antibodydescribed herein, numbered according to the EU numbering system, can bereplaced with a different amino acid residue such that the antibody hasaltered C1q binding and/or reduced or abolished complement dependentcytotoxicity (CDC). This approach is described in further detail in U.S.Pat. No. 6,194,551 (Idusogie et al). In some embodiments, one or moreamino acid residues within amino acid positions 231 to 238 in theN-terminal region of the CH2 domain of an antibody described herein arealtered to thereby alter the ability of the antibody to fix complement.This approach is described further in International Publication No. WO94/29351. In certain embodiments, the Fc region of an antibody describedherein is modified to increase the ability of the antibody to mediateantibody dependent cellular cytotoxicity (ADCC) and/or to increase theaffinity of the antibody for an Fcγ receptor by mutating one or moreamino acids (e.g., introducing amino acid substitutions) at thefollowing positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265,267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292,293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322,324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360,373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437,438, or 439, numbered according to the EU numbering system. Thisapproach is described further in International Publication No. WO00/42072.

In certain embodiments, an antibody described herein comprises theconstant domain of an IgG₁ with a mutation (e.g., substitution) atposition 267, 328, or a combination thereof, numbered according to theEU numbering system. In certain embodiments, an antibody describedherein comprises the constant domain of an IgG₁ with a mutation (e.g.,substitution) selected from the group consisting of S267E, L328F, and acombination thereof, numbered according to the EU numbering system. Incertain embodiments, an antibody described herein comprises the constantdomain of an IgG₁ with a S267E/L328F mutation (e.g., substitution),numbered according to the EU numbering system. In certain embodiments,an antibody described herein comprising the constant domain of an IgG₁with a S267E/L328F mutation (e.g., substitution) has an increasedbinding affinity for FcγRIIA, FcγRIIB, or FcγRIIA and FcγRIIB, numberedaccording to the EU numbering system.

In certain embodiments, an antibody described herein comprises theconstant region of an IgG₄ antibody and the serine at amino acid residue228 of the heavy chain, numbered according to the EU numbering system,is substituted for proline.

In certain embodiments, an antibody described herein comprises theconstant region of an IgG₂ antibody and the cysteine at amino acidresidue 127 of the heavy chain, numbered according to Kabat, issubstituted for serine.

Antibodies with reduced fucose content have been reported to have anincreased affinity for Fc receptors, such as, e.g., FcγRIIIa.Accordingly, in certain embodiments, the antibodies described hereinhave reduced fucose content or no fucose content. Such antibodies can beproduced using techniques known to one skilled in the art. For example,the antibodies can be expressed in cells deficient or lacking theability of fucosylation. In a specific example, cell lines with aknockout of both alleles of α1,6-fucosyltransferase can be used toproduce antibodies with reduced fucose content. The Potelligent® system(Lonza) is an example of such a system that can be used to produceantibodies with reduced fucose content. Alternatively, antibodies withreduced fucose content or no fucose content can be produced by, e.g.:(i) culturing cells under conditions which prevent or reducefucosylation; (ii) posttranslational removal of fucose (e.g., with afucosidase enzyme); (iii) post-translational addition of the desiredcarbohydrate, e.g., after recombinant expression of a non-glycosylatedglycoprotein; or (iv) purification of the glycoprotein so as to selectfor antibodies thereof which are not fucsoylated. See, e.g., Longmore GD & Schachter H (1982) Carbohydr Res 100: 365-92 and Imai-Nishiya H etal., (2007) BMC Biotechnol. 7: 84 for methods for producing antibodiesthereof with no fucose content or reduced fucose content.

Engineered glycoforms may be useful for a variety of purposes, includingbut not limited to enhancing or reducing effector function. Methods forgenerating engineered glycoforms in an antibody described herein includebut are not limited to those disclosed, e.g., in Umalia P et al., (1999)Nat Biotechnol 17: 176-180; Davies J et al., (2001) Biotechnol Bioeng74: 288-294; Shields R L et al., (2002) J Biol Chem 277: 26733-26740;Shinkawa T et al., (2003) J Biol Chem 278: 3466-3473; Niwa R et al.,(2004) Clin Cancer Res 1: 6248-6255; Presta L G et al., (2002) BiochemSoc Trans 30: 487-490; Kanda Y et al., (2007) Glycobiology 17: 104-118;U.S. Pat. Nos. 6,602,684; 6,946,292; and 7,214,775; U.S. PatentPublication Nos. US 2007/0248600; 2007/0178551; 2008/0060092; and2006/0253928; International Publication Nos. WO 00/61739; WO 01/292246;WO 02/311140; and WO 02/30954; Potillegent™ technology (Biowa, Inc.Princeton, N.J.); and GlycoMAb® glycosylation engineering technology(Glycart biotechnology AG, Zurich, Switzerland). See also, e.g., FerraraC et al., (2006) Biotechnol Bioeng 93: 851-861; InternationalPublication Nos. WO 07/039818; WO 12/130831; WO 99/054342; WO 03/011878;and WO 04/065540.

In certain embodiments, the technology used to engineer the Fc domain ofan antibody described herein is the Xmab® Technology of Xencor(Monrovia, CA). See, e.g., U.S. Pat. Nos. 8,367,805; 8,039,592;8,124,731; 8,188,231; U.S. Patent Publication No. 2006/0235208;International Publication Nos. WO 05/077981; WO 11/097527; and RichardsJ O et al., (2008) Mol Cancer Ther 7: 2517-2527.

In certain embodiments, amino acid residues in the constant region of anantibody described herein in the positions corresponding to positionsL234, L235, and D265 in a human IgG1 heavy chain, numbered according tothe EU numbering system, are not L, L, and D, respectively. Thisapproach is described in detail in International Publication No. WO14/108483. In a particular embodiment, the amino acids corresponding topositions L234, L235, and D265 in a human IgG1 heavy chain are F, E, andA; or A, A, and A, respectively, numbered according to the EU numberingsystem.

In certain embodiments, one, two, or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of an antibodydescribed herein (e.g., CH2 domain (residues 231-340 of human IgG₁)and/or CH3 domain (residues 341-447 of human IgG₁) and/or the hingeregion, with numbering according to the EU numbering system, e.g., toalter one or more functional properties of the antibody, such as serumhalf-life, complement fixation, Fc receptor binding and/orantigen-dependent cellular cytotoxicity.

In certain embodiments, one, two, or more mutations (e.g., amino acidsubstitutions) are introduced into the hinge region of the Fc region(CH1 domain) such that the number of cysteine residues in the hingeregion are altered (e.g., increased or decreased) as described in, e.g.,U.S. Pat. No. 5,677,425. The number of cysteine residues in the hingeregion of the CH1 domain may be altered to, e.g., facilitate assembly ofthe light and heavy chains, or to alter (e.g., increase or decrease) thestability of the antibody.

In certain embodiments, a multispecific (e.g., bispecific) antibody,which immunospecifically binds to GITR and OX40 (e.g., human GITR andOX40), increases GITR and/or OX40 (e.g., human GITR and/or OX40)activity by at least about 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold as assessed bymethods described herein and/or known to one of skill in the art,relative to GITR and/or OX40 (e.g., human GITR and/or OX40) activitywithout any antibody or with an unrelated antibody (e.g., an antibodythat does not immunospecifically bind to GITR or OX40). For instance, anantibody that binds to GITR and OX40, e.g., an antibody that binds toGITR and OX40 and comprises a combination of CDR sequences specifiedherein, a VH and/or VL sequence having at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99% or 100% sequence identity with VH and/or VL sequencesspecified herein, or heavy and/or light chains specified herein, can,increase GITR and/or OX40 (e.g., human GITR and/or OX40) activity by atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% as assessed by methodsdescribed herein and/or known to one of skill in the art, relative toGITR and/or OX40 (e.g., human GITR and/or OX40) activity without anyantibody or with an unrelated antibody (e.g., an antibody that does notimmunospecifically bind to GITR or OX40). Non-limiting examples of GITRand/or OX40 (e.g., human GITR and/or OX40) activity can include GITRand/or OX40 (e.g., human GITR and/or OX40) signaling, cellproliferation, cell survival, and cytokine production (e.g., IL-2,TNF-α, IFN-γ, IL-4, IL-10, and/or IL-13).

As provided herein, multispecific antibodies (e.g., bispecificantibodies) that bind to GITR and/or OX40 can agonize GITR and/or OX40function, for example, by stimulating IL-2 release in SEA assay, e.g.,as exemplified in the Examples, infra. For instance, an antibody thatbinds to GITR and OX40, e.g., an antibody that binds to GITR and OX40and comprises a combination of CDR sequences specified herein, a VHand/or VL sequence having at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99% or100% sequence identity with VH and/or VL sequences specified herein, orheavy and/or light chains specified herein, can, in combination withStaphylococcus Enterotoxin A (SEA) (e.g., 100 ng/ml), induce IL-2production in, e.g., PBMCs upon stimulation for, e.g., 5 days at, e.g.,37° C., 5% CO₂, and 97% humidity, as measured by, e.g.,electrochemiluminescence. In some embodiments, the IL-2 production is asubstantially increasing function of antibody concentrations between,e.g., 0.08 μg/ml and 20 μg/ml, 0.25 μg/ml and 20 μg/ml, 0.74 μg/ml and20 μg/ml, 2.2 μg/ml and 20 μg/ml, or 6.7 μg/ml and 20 μg/ml. In certainembodiments, an antibody that binds to GITR and OX40, e.g., an antibodythat binds to GITR and OX40 and comprises a combination of CDR sequencesspecified herein, a VH and/or VL sequence having at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99% or 100% sequence identity with VH and/or VL sequencesspecified herein, or heavy and/or light chains specified herein, can, incombination with Staphylococcus Enterotoxin A (SEA), induce IL-2production in, e.g., PBMCs, wherein the IL-2 production is asubstantially increasing function of antibody concentrations between,e.g., 0.08 μg/ml and 20 μg/ml, 0.25 μg/ml and 20 μg/ml, 0.74 μg/ml and20 μg/ml, 2.2 μg/ml and 20 μg/ml, or 6.7 μg/ml and 20 μg/ml as assessedin, e.g., an assay comprising the following steps: (a) culturing thePBMCs (e.g., 10⁵ cells in a well) in the absence or presence of varyingconcentrations (e.g., 20, 46.7, 2.2, 0.74, 0.25, and 0.08m/1111) of theantibody and, e.g., 100 ng/ml of SEA for, e.g., 5 days at, e.g., 37° C.,5% CO₂, and 97% humidity; and (b) collecting clarified supernatant andmeasuring the titer of IL-2 by, e.g., electrochemiluminescence.

In certain embodiments, an antibody that binds to GITR and OX40, e.g.,an antibody that binds to GITR and OX40 and comprises a combination ofCDR sequences specified herein, a VH and/or VL sequence having at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, at least 99% or 100% sequence identity with VH and/orVL sequences specified herein, or heavy and/or light chains specifiedherein, can, in combination with Staphylococcus Enterotoxin A (SEA)(e.g., 100 ng/ml), induce IL-2 production in, e.g., PBMCs uponstimulation for, e.g., 5 days at, e.g., 37° C., 5% CO₂, and 97%humidity, as measured by, e.g., electrochemiluminescence. In someembodiments, the IL-2 production shows a sigmoidal dose response curvewhen the antibody is between, e.g., 0.08 μg/ml and 20 μg/ml, 0.25 μg/mland 20 μg/ml, 0.74 μg/ml and 20 μg/ml, 2.2 μg/ml and 20 μg/ml, or 6.7μg/ml and 20 μg/ml. In certain embodiments, an antibody that binds toGITR and OX40, e.g., an antibody that binds to GITR and OX40 andcomprises a combination of CDR sequences specified herein, a VH and/orVL sequence having at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 98%, at least 99% or 100%sequence identity with VH and/or VL sequences specified herein, or heavyand/or light chains specified herein, can, in combination withStaphylococcus Enterotoxin A (SEA), induce IL-2 production in, e.g.,PBMCs, wherein the IL-2 production shows a sigmoidal dose response curvewhen the antibody is between, e.g., 0.08 μg/ml and 20 μg/ml, 0.25 μg/mland 20 μg/ml, 0.74 μg/ml and 20 μg/ml, 2.2 μg/ml and 20 μg/ml, or 6.7μg/ml and 20 μg/ml as assessed in, e.g., an assay comprising thefollowing steps: (a) culturing the PBMCs (e.g., 10⁵ cells in a well) inthe absence or presence of varying concentrations (e.g., 20, 6.7, 2.2,0.74, 0.25, and 0.08m/1111) of the antibody and, e.g., 100 ng/ml of SEAfor, e.g., 5 days at, e.g., 37° C., 5% CO₂, and 97% humidity; and (b)collecting clarified supernatant and measuring the titer of IL-2 by,e.g., electrochemiluminescence. In certain embodiments, an antibody thatbinds to GITR and OX40, e.g., an antibody that binds to GITR and OX40and comprises a combination of CDR sequences specified herein, a VHand/or VL sequence having at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99% or100% sequence identity with VH and/or VL sequences specified herein, orheavy and/or light chains specified herein, can, in combination withStaphylococcus Enterotoxin A (SEA) (e.g., 100 ng/ml), induce IL-2production in, e.g., PBMCs upon stimulation for, e.g., 5 days at, e.g.,37° C., 5% CO₂, and 97% humidity, as measured by, e.g.,electrochemiluminescence, wherein the IL-2 production is a substantiallyincreasing function of antibody concentrations between, e.g., 0.08 μg/mland 20 μg/ml, 0.25 μg/ml and 20 μg/ml, 0.74 μg/ml and 20 μg/ml, 2.2μg/ml and 20 μg/ml, or 6.7 μg/ml and 20 μg/ml. In certain embodiments,an antibody that binds to GITR and OX40, e.g., an antibody that binds toGITR and OX40 and comprises a combination of CDR sequences specifiedherein, a VH and/or VL sequence having at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99% or 100% sequence identity with VH and/or VL sequencesspecified herein, or heavy and/or light chains specified herein, can, incombination with Staphylococcus Enterotoxin A (SEA), induce IL-2production in, e.g., PBMCs, wherein the IL-2 production is asubstantially increasing function of antibody concentrations between,e.g., 0.08 μg/ml and 20 μg/ml, 0.25 μg/ml and 20 μg/ml, 0.74 μg/ml and20 μg/ml, 2.2 μg/ml and 20 μg/ml, or 6.7 μg/ml and 20 μg/ml, as assessedin, e.g., an assay comprising the following steps: (a) culturing thePBMCs (e.g., 10⁵ cells in a well) in the absence or presence of varyingconcentrations (e.g., 20, 6.7, 2.2, 0.74, 0.25, and 0.08 μg/ml) of theantibody and, e.g., 100 ng/ml of SEA for, e.g., 5 days at, e.g., 37° C.,5% CO₂, and 97% humidity; and (b) collecting clarified supernatant andmeasuring the titer of IL-2 by, e.g., electrochemiluminescence.

In a specific aspect, provided herein are multispecific (e.g.,bispecific) antagonistic antibodies, which immunospecifically bind toGITR and OX40 (e.g., human GITR and OX40).

In a specific aspect, a multispecific (e.g., bispecific) antibody asdescribed herein, which immunospecifically binds to GITR and OX40 (e.g.,human GITR and OX40), comprises a human immunoglobulin IgG₁ heavy chainconstant region, wherein the amino acid sequence of the IgG₁ heavy chainconstant region comprises a mutation selected from the group consistingof: N297A, D265A, L234F, L235E, N297Q, and P331S, numbered according tothe EU numbering system. In certain embodiments, the mutation is N297Aor D265 A, numbered according to the EU numbering system. In certainembodiments the mutation is L234F and L235E, numbered according to theEU numbering system. In certain embodiments, the mutation is L234F,L234E, and D265A, numbered according to the EU numbering system. Incertain embodiments, the mutation is L234F, L234E, and N297Q, numberedaccording to the EU numbering system. In certain embodiments, themutation is L234F, L235E, and P331S, numbered according to the EUnumbering system. In certain embodiments, the mutation is D265A andN297Q, numbered according to the EU numbering system. In certainembodiments, the mutation is L234F, L235E, D265A, N297Q, and P331S,numbered according to the EU numbering system. In a specific aspect, amultispecific (e.g., bispecific) antibody as described herein, whichimmunospecifically binds to GITR and OX40 (e.g., human GITR and OX40),comprises a human immunoglobulin IgG₁ heavy chain constant region,wherein the amino acid sequence of the IgG₁ heavy chain constant regioncomprises a mutation selected from the group consisting of D265A, P329A,and a combination thereof, numbered according to the EU numberingsystem. In certain embodiments, the antibody is antagonistic.

In certain embodiments, an antagonist multispecific (e.g., bispecific)antibody described herein, which immunospecifically binds to GITR andOX40 (e.g., human GITR and OX40), decreases GITR and/or OX40 (e.g.,human GITR and/or OX40) activity by at least about 1.2 fold, 1.3 fold,1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold,30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, fold, or 100 foldas assessed by methods described herein and/or known to one of skill inthe art, relative to GITR and/or OX40 (e.g., human GITR and/or OX40)activity without any antibody or with an unrelated antibody (e.g., anantibody that does not immunospecifically bind to GITR or OX40). Incertain embodiments, an antagonist multispecific (e.g., bispecific)antibody described herein, which immunospecifically binds to GITR and/orOX40 (e.g., human GITR and/or OX40), decreases GITR and/or OX40 (e.g.,human GITR and/or OX40) activity by at least 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, or 99% as assessed by methods described herein and/or known to oneof skill in the art, relative to GITR and/or OX40 (e.g., human GITRand/or OX40) activity without any antibody or with an unrelated antibody(e.g., an antibody that does not immunospecifically bind to GITR orOX40). Non-limiting examples of GITR and/or OX40 (e.g., human GITRand/or OX40) activity can include GITR and/or OX40 (e.g., human GITRand/or OX40) signaling, cell proliferation, cell survival, and cytokineproduction (e.g., IL-2, TNF-α, IFN-γ, IL-4, IL-10, and/or IL-13). Inspecific embodiments, GITR and/or OX40 activity is assessed as describedin the Examples, infra.

As provided herein, antagonist multispecific antibodies (e.g.,bispecific antibodies) that bind to GITR and/or OX40 can antagonize GITRand/or OX40 function, for example, by neutralize GITRL-inducedsignaling, e.g., as exemplified in the Examples, infra. For instance, anantagonist antibody that binds to GITR and OX40, e.g., an antagonistantibody that binds to GITR and OX40 and comprises a combination of CDRsequences specified herein, a VH and/or VL sequence having at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99% or 100% sequence identity with VH and/or VLsequences specified herein, or heavy and/or light chains specifiedherein, can neutralize GITRL-induced signaling as measured by, e.g., aluciferase assay. In certain embodiments, an antagonist antibody thatbinds to GITR and OX40, e.g., an antagonist antibody that binds to GITRand OX40 and comprises a combination of CDR sequences specified herein,a VH and/or VL sequence having at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or100% sequence identity with VH and/or VL sequences specified herein, orheavy and/or light chains specified herein, can neutralize GITRL-inducedsignaling as assessed in, e.g., a luciferase assay comprising thefollowing steps: (a) culturing Jurkat-huGITR-NF-κB-luciferase cells inthe absence or presence of varying concentrations of the antibody (e.g.,12-point dose titration, 0.05-10,000 ng/ml) and trimeric GITRL for 2hours in RPMI media, supplemented with 10% heat-inactivated FBS, at 37°C. and 5% CO₂ and (b) detecting luciferase activity.

In certain embodiments, antagonist multispecific antibodies (e.g.,bispecific antibodies) described herein that bind to GITR and/or OX40block the interaction of GITR and/or OX40 with GITRL and/or OX40L (e.g.,blocks the binding of GITRL and GITR and/or OX40L and OX40 to oneanother). In certain embodiments, antagonist multispecific antibodies(e.g., bispecific antibodies) described herein that bind to GITR and/orOX40 decrease GITR and/or OX40 activity (e.g., GITR and/or OX40signaling) induced by GITRL and/or OX40L. In certain embodiments,antagonist multispecific antibodies (e.g., bispecific antibodies)described herein that bind to GITR and/or OX40 suppress T cellproliferation. In certain embodiments, antagonist multispecificantibodies (e.g., bispecific antibodies) described herein that bind toGITR and/or OX40 suppress production of cytokines (e.g., IL-2, TNFα,IFNγ, IL-4, IL-10, IL-13, or a combination thereof).

An antibody provided herein that binds to GITR and/or OX40 can be fusedor conjugated (e.g., covalently or noncovalently linked) to a detectablelabel or substance. Examples of detectable labels or substances includeenzyme labels, such as, glucose oxidase; radioisotopes, such as iodine(¹²⁵I, ¹²¹I) carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹²¹In),and technetium (⁹⁹Tc); luminescent labels, such as luminol; andfluorescent labels, such as fluorescein and rhodamine, and biotin. Suchlabeled antibodies can be used to detect OX40 (e.g., human OX40)protein. See, e.g., Section 7.5.2, infra.

7.2.1 OX40 Antigen-Binding Domains

In a particular embodiment, an OX40 antigen-binding domain describedherein, which specifically binds to OX40 (e.g., human OX40), comprises acomprising a light chain variable region (VL) comprising:

-   -   (a) a VL CDR1 comprising, consisting of, or consisting        essentially of the amino acid sequence RSSQSLLHSNGYNYLD (SEQ ID        NO:50),    -   (b) a VL CDR2 comprising, consisting of, or consisting        essentially of the amino acid sequence LGSNRAS (SEQ ID NO:51),        and    -   (c) a VL CDR3 comprising, consisting of, or consisting        essentially of the amino acid sequence MQALQTPLT (SEQ ID NO:52)        or MQALQTPLT (SEQ ID NO:53), as shown in Table 4. In some        embodiments, the OX40 antigen-binding domain comprises the VL        framework regions described herein.

In another embodiment, an antigen-binding domain described herein, whichspecifically binds to OX40 (e.g., human OX40), comprises a heavy chainvariable region (VH) comprising:

-   -   (a) a VH CDR1 comprising, consisting of, or consisting        essentially of the amino acid sequence GSAMH (SEQ ID NO:47),    -   (b) a VH CDR2 comprising, consisting of, or consisting        essentially of the amino acid sequence RIRSKANSYATAYAASVKG (SEQ        ID NO:48), and    -   (c) a VH CDR3 comprising, consisting of, or consisting        essentially of the amino acid sequence GIYDSSGYDY (SEQ ID        NO:49), as shown in Table 4.

In some embodiments, the OX40 antigen-binding domain comprises the VHframeworks described herein. In specific embodiments, the OX40antigen-binding domain comprises the VH framework regions of an antibodydescribed herein.

TABLE 4 VL CDR amino acid sequences of anti-OX40 antibodies * VL CDR1VL CDR2 VL CDR3 Antibody (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) pab1949wRSSQSLLHSNGYNYLD LGSNRAS (51) MQALQTPLT (50) (53) pab2049wRSSQSLLHSNGYNYLD LGSNRAS (51) MQGSKWPLT (50) (52) * The VL CDRs in Table4 are determined according to Kabat.

TABLE 5 VH CDR amino acid sequences of anti-OX40 antibodies * VH CDR1VH CDR2 VH CDR3 Antibody (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) pab1949wGSAMH (47) RIRSKANSYATAYAASVKG GIYDSSGYDY (48) (49) pab2049w GSAMH (47)RIRSKANSYATAYAASVKG GIYDSSGYDY (48) (49) * The VH CDRs in Table 5 aredetermined according to Kabat.

In certain embodiments, provided herein is an antigen-binding domainwhich specifically binds to OX40 (e.g., human OX40) and comprises lightchain variable region (VL) CDRs and heavy chain variable region (VH)CDRs of pab1949w, or pab2049w, for example as set forth in Tables 4 and5 (i.e., SEQ ID NOs:47-52 or SEQ ID NOs:47-51 and 53).

In certain embodiments, an OX40 antigen-binding domain comprises a lightchain variable framework region that is derived from a human IGKV2-28germline sequence (e.g., IGKV2-28*01, e.g., having amino acid sequenceof SEQ ID NO:58).

In certain embodiments, the OX40 antigen-binding domain comprises aheavy chain variable framework region that is derived from humanIGHV3-73 germline sequence (e.g., IGHV3-73*01, e.g., having amino acidsequence of SEQ ID NO:57).

In a specific embodiment, an antigen-binding domain that specificallybinds to OX40 (e.g., human OX40) comprises a VL domain comprising theamino acid sequence of SEQ ID NO:55 or 56. In a specific embodiment, anantigen-binding domain that specifically binds to OX40 (e.g., humanOX40) comprises a VL domain consisting of or consisting essentially ofthe amino acid sequence of SEQ ID NO:55 or 56.

In certain embodiments, an antigen-binding domain that specificallybinds to OX40 (e.g., human OX40) comprises a VH domain comprising theamino acid sequence of SEQ ID NO:54. In some embodiments, anantigen-binding domain that specifically binds to OX40 (e.g., humanOX40) comprises a VH domain consisting of or consisting essentially ofthe amino acid sequence of SEQ ID NO:54.

In certain embodiments, an antigen-binding domain that specificallybinds to OX40 (e.g., human OX40) comprises a VH domain and a VL domain,wherein the VH domain and the VL domain comprise the amino acidsequences of SEQ ID NO:54 and SEQ ID NO:55 or 56, respectively. Incertain embodiments, an antigen-binding domain that specifically bindsto OX40 (e.g., human OX40) comprises a VH domain and a VL domain,wherein the VH domain and the VL domain consist of or consistessentially of the amino acid sequences of SEQ ID NO:54 and SEQ ID NO:55or 56, respectively, e.g., as shown in Table 6.

TABLE 6 VH and VL sequences of exemplary anti-OX40 antibodies AntibodyVH (SEQ ID NO:) VL (SEQ ID NO:) pab2049w 54 55 pab1949w 54 56In specific aspects, provided herein is an antigen-binding domaincomprising a light chain and heavy chain, e.g., a separate light chainand heavy chain. With respect to the light chain, in a specificembodiment, the light chain of an antigen-binding domain describedherein is a kappa light chain. In another specific embodiment, the lightchain of an antigen-binding domain described herein is a lambda lightchain. In yet another specific embodiment, the light chain of anantigen-binding domain described herein is a human kappa light chain ora human lambda light chain. In a particular embodiment, anantigen-binding domain described herein, which immunospecifically bindsto an OX40 polypeptide (e.g., human OX40) comprises a light chainwherein the amino acid sequence of the VL domain comprises the sequenceset forth in SEQ ID NO:55 or 56, and wherein the constant region of thelight chain comprises the amino acid sequence of a human kappa lightchain constant region. In another particular embodiment, anantigen-binding domain described herein, which immunospecifically bindsto OX40 (e.g., human OX40) comprises a light chain wherein the aminoacid sequence of the VL domain comprises the sequence set forth in SEQID NO:55 or 56 and wherein the constant region of the light chaincomprises the amino acid sequence of a human lambda light chain constantregion. In a specific embodiment, an antigen-binding domain describedherein, which immunospecifically binds to OX40 (e.g., human OX40)comprises a light chain wherein the amino acid sequence of the VL domaincomprises the sequence set forth in SEQ ID NO:55 or 56 and wherein theconstant region of the light chain comprises the amino acid sequence ofa human kappa or lambda light chain constant region. Non-limitingexamples of human constant region sequences have been described in theart, e.g., see U.S. Pat. No. 5,693,780 and Kabat E A et al., (1991)supra.

In a particular embodiment, an antigen-binding domain described herein,which specifically binds to OX40 (e.g., human OX40) comprises a lightchain comprising the amino acid sequence set forth in SEQ ID NO:67 or69.

With respect to the heavy chain, in a specific embodiment, the heavychain of an antigen-binding domain described herein can be an alpha (α),delta (δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In anotherspecific embodiment, the heavy chain of an antigen-binding domaindescribed can comprise a human alpha (α), delta (δ), epsilon (ε), gamma(γ) or mu (μ) heavy chain. In a particular embodiment, anantigen-binding domain described herein, which immunospecifically bindsto OX40 (e.g., human OX40), comprises a heavy chain wherein the aminoacid sequence of the VH domain can comprise the sequence set forth inSEQ ID NO:54 and wherein the constant region of the heavy chaincomprises the amino acid sequence of a human gamma (γ) heavy chainconstant region. In a specific embodiment, an antigen-binding domaindescribed herein, which specifically binds to OX40 (e.g., human OX40),comprises a heavy chain wherein the amino acid sequence of the VH domaincomprises the sequence set forth in SEQ ID NO:54, and wherein theconstant region of the heavy chain comprises the amino acid of a humanheavy chain described herein or known in the art. Non-limiting examplesof human constant region sequences have been described in the art, e.g.,see U.S. Pat. No. 5,693,780 and Kabat E A et al., (1991) supra.

In a particular embodiment, an antigen-binding domain described herein,which specifically binds to OX40 (e.g., human OX40), comprises a heavychain comprising the amino acid sequence set forth in SEQ ID NO:61. Inanother embodiment, an antigen-binding domain described herein, whichspecifically binds to OX40 (e.g., human OX40), comprises a heavy chaincomprising the amino acid sequence set forth in SEQ ID NO:62. In anotherembodiment, an antigen-binding domain described herein, whichspecifically binds to OX40 (e.g., human OX40), comprises a heavy chaincomprising the amino acid sequence set forth in SEQ ID NO:63. In anotherembodiment, an antigen-binding domain described herein, whichspecifically binds to OX40 (e.g., human OX40), comprises a heavy chaincomprising the amino acid sequence set forth in SEQ ID NO:64. In anotherembodiment, an antigen-binding domain described herein, whichspecifically binds to OX40 (e.g., human OX40), comprises a heavy chaincomprising the amino acid sequence set forth in SEQ ID NO:65. In anotherembodiment, an antigen-binding domain described herein, whichspecifically binds to OX40 (e.g., human OX40), comprises a heavy chaincomprising the amino acid sequence set forth in SEQ ID NO:71.

In a specific embodiment, an antigen-binding domain described herein,which immunospecifically binds to OX40 (e.g., human OX40), comprises aVL domain and a VH domain comprising any amino acid sequences describedherein, wherein the constant regions comprise the amino acid sequencesof the constant regions of an IgG, IgE, IgM, IgD, IgA, or IgYimmunoglobulin molecule, or a human IgG, IgE, IgM, IgD, IgA, or IgYimmunoglobulin molecule. In another specific embodiment, anantigen-binding domain described herein, which immunospecifically bindsto OX40 (e.g., human OX40) comprises a VL domain and a VH domaincomprising any amino acid sequences described herein, wherein theconstant regions comprise the amino acid sequences of the constantregions of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule,any class (e.g., IgG₁,IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂), or any subclass(e.g., IgG₂a and IgG_(2b)) of immunoglobulin molecule. In a particularembodiment, the constant regions comprise the amino acid sequences ofthe constant regions of a human IgG, IgE, IgM, IgD, IgA, or IgYimmunoglobulin molecule, any class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁,and IgA₂), or any subclass (e.g., IgG_(2a) and IgG_(2b)) ofimmunoglobulin molecule.

In another specific embodiment, an antigen-binding domain describedherein, which immunospecifically binds to OX40 (e.g., human OX40),comprises a VL domain and a VH domain comprising any amino acidsequences described herein, wherein the constant regions comprise theamino acid sequences of the constant regions of a human IgG₁ (e.g.,allotypes G1m3, G1m17,1 or Glm17,1,2), human IgG₂, or human IgG₄. In aparticular embodiment, an antigen-binding domain described herein, whichimmunospecifically binds to OX40 (e.g., human OX40), comprises a VLdomain and a VH domain comprising any amino acid sequences describedherein, wherein the constant regions comprise the amino acid sequencesof the constant region of a human IgG₁ (allotype Glm3). Non-limitingexamples of human constant regions are described in the art, e.g., seeKabat E A et al., (1991) supra.

In another embodiment, an antigen-binding domain described herein, whichspecifically binds to OX40 (e.g., human OX40), comprises a light chaincomprising the amino acid sequence set forth in SEQ ID NO:67 and a heavychain comprising the amino acid sequence set forth in SEQ ID NO:61, 62,63, 64, 65, or 71. In another embodiment, an antigen-binding domaindescribed herein, which specifically binds to OX40 (e.g., human OX40),comprises a light chain comprising the amino acid sequence set forth inSEQ ID NO:69 and a heavy chain comprising the amino acid sequence setforth in SEQ ID NO:61, 62, 63, 64, 65, or 71.

In certain embodiments, an antigen-binding domain described herein,which immunospecifically binds to OX40 (e.g., human OX40), comprises aVL domain having at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, or at least 98% sequence identity to theamino acid sequence of the VL domain of pab1949w or pab2049w (i.e., SEQID NO:55 or 56), e.g., wherein the antigen-binding domain comprises VLCDRs that are identical to the VL CDRs of pab1949w or pab2049w.

In certain embodiments, an antigen-binding domain described herein,which immunospecifically binds to OX40 (e.g., human OX40), comprises aVH domain having at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, or at least 98% sequence identity to theamino acid sequence of the VH domain of pab1949w or pab2049w (i.e., SEQID NO:54), e.g., wherein the antigen-binding domain comprises VH CDRsthat are identical to the VH CDRs of pab1949w or pab2049w.

In certain embodiments, an antigen-binding domain described herein,which immunospecifically binds to OX40 (e.g., human OX40), comprises:(i) a VL domain having at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 98% sequence identityto the amino acid sequence of the VL domain of pab1949w or pab2049w(i.e., SEQ ID NO:55 or 56); and (ii) a VH domain having at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 98% sequence identity to the amino acid sequence of the VH domainof pab1949w or pab2049w (i.e., SEQ ID NO:54), e.g., wherein the antibodycomprises VL CDRs and VH CDRs that are identical to the VL CDRs and VHCDRs of pab1949w or pab2049w.

In specific aspects, provided herein is an antigen-binding domain whichcompetes (e.g., in a dose dependent manner) for specific binding to OX40(e.g., human OX40), with an antigen-binding domain comprising a VLdomain having the amino acid sequence set forth in SEQ ID NO:55 or 56,and a VH domain having the amino acid sequence set for the in SEQ IDNO:54.

In a specific embodiment, an antigen-binding domain described herein isone that is competitively blocked (e.g., in a dose dependent manner) byan antigen-binding domain comprising a VL domain having the amino acidsequence set forth in SEQ ID NO:55 or 56 and a VH domain having theamino acid sequence set forth in SEQ ID NO:54 for specific binding toOX40 (e.g., human OX40).

Assays known to one of skill in the art or described herein (e.g., X-raycrystallography, hydrogen/deuterium exchange coupled with massspectrometry (e.g., liquid chromatography electrospray massspectrometry), alanine scanning, ELISA assays, etc.) can be used todetermine if two antibodies bind to the same epitope.

In a specific embodiment, an antigen-binding domain described hereinimmunospecifically binds to the same epitope as that bound by pab1949wor pab2049w or an epitope that overlaps the epitope.

In a specific aspect, the binding between an antigen-binding domaindescribed herein and a variant OX40 is substantially weakened relativeto the binding between the antigen-binding domain and a human OX40sequence of SEQ ID NO:72, wherein the variant OX40 comprises thesequence of SEQ ID NO:72 except for an amino acid mutation (e.g.,substitution) selected from the group consisting of: N60A, R62A, R80A,L88A, P93A, P99A, P115A, and a combination thereof or selected from thegroup consisting of N60A, R62A, R80A, L88A, and P93A, numbered accordingto SEQ ID NO: 72.

In some embodiments, the variant OX40 comprises the sequence of SEQ IDNO:72 except for any one mutation selected from the group consisting of:N60A, R62A, R80A, L88A, P93A, P99A, and P115A or selected from the groupconsisting of N60A, R62A, R80A, L88A, and P93A, numbered according toSEQ ID NO: 72. In some embodiments, the variant OX40 comprises thesequence of SEQ ID NO: 72 except for any two, three, four, five, six, orseven mutations selected from the group consisting of: W58A, N60A, R62A,R80A, L88A, P93A, P99A, and P115A or selected from the group consistingof N60A, R62A, R80A, L88A, and P93A, numbered according to SEQ ID NO:72. In some embodiments, the variant OX40 comprises the sequence of SEQID NO:72 except for the amino acid mutations W58A, N60A, R62A, R80A,L88A, P93A, P99A, and P115A or except for the amino acid mutations ofN60A, R62A, R80A, L88A, and P93A, numbered according to SEQ ID NO: 72.

In a specific aspect, an antigen-binding domain described herein bindsto an epitope of a human OX40 sequence comprising, consistingessentially of, or consisting of a residue of SEQ ID NO:72 selected fromthe group consisting of: 58, 60, 62, 80, 88, 93, 99, 115, and acombination thereof or elected from the group consisting of: 60, 62, 80,88, 93, and a combination thereof. In some embodiments, the epitopecomprises, consists of, or consists essentially of any one residue, orany two, three, four, five, six, or seven residues, selected from thegroup consisting of: 58, 60, 62, 80, 88, 93, 99, and 115 of SEQ ID NO:72or selected from the group consisting of: 60, 62, 80, 88, and 93 of SEQID NO:72. In some embodiments, the epitope comprises, consistsessentially of, or consists of residues 58, 60, 62, 80, 88, 93, 99, and115 of SEQ ID NO:72 or comprises residues 60, 62, 80, 88, and 93 of SEQID NO:72.

In a specific embodiment, an antigen-binding domain described hereinbinds to an epitope of SEQ ID NO:72 comprising, consisting essentiallyof, or consisting of a residue selected from the group consisting of:58, 60, 62, 80, 88, 93, 99, 115, and a combination thereof or an epitopeof SEQ ID NO:72 comprising, consisting essentially of, or consisting ofa residue selected from the group consisting of: 60, 62, 80, 88, 93, anda combination thereof. In some embodiments, the epitope comprises anyone residue, or any two, three, four, five, six, or seven residues,selected from the group consisting of: 58, 60, 62, 80, 88, 93, 99, and115 of SEQ ID NO:72 or selected from the group consisting of: 60, 62,80, 88, and 93 of SEQ ID NO:72. In some embodiments, the epitopecomprises, consists of, or consists essentially of residues 58, 60, 62,80, 88, 93, 99, and 115 of SEQ ID NO:72 or comprises residues 60, 62,80, 88, and 93 of SEQ ID NO:72.

In a specific aspect, an antigen-binding domain described herein bindsto at least one residue of SEQ ID NO:72 selected from the groupconsisting of: 58, 60, 62, 80, 88, 93, 99, 115, and a combinationthereof or selected from the group consisting of: 60, 62, 80, 88, 93,and a combination thereof. In some embodiments, an antigen-bindingdomain described herein binds to any one residue, or any two, three,four, five, six, or seven residues, selected from the group consistingof: 58, 60, 62, 80, 88, 93, 99, and 115 of SEQ ID NO:72 or selected fromthe group consisting of: 60, 62, 80, 88, and 93 of SEQ ID NO:72. In someembodiments, an antigen-binding domain described herein binds toresidues 58, 60, 62, 80, 88, 93, 99, and 115 of SEQ ID NO:72. In someembodiments, an antigen-binding domain described herein binds toresidues 60, 62, 80, 88, and 93 of SEQ ID NO:72.

In a specific aspect, an antigen-binding domain described hereinexhibits, as compared to binding to a human OX40 sequence of SEQ IDNO:72, reduced or absent binding to a protein identical to SEQ ID NO:72except for the presence of an amino acid mutation (e.g., substitution)selected from the group consisting of: N60A, R62A, R80A, L88A, P93A,P99A, P115A, and a combination thereof or selected from the groupconsisting of: N60A, R62A, R80A, L88A, P93A, and a combination thereof,numbered according to SEQ ID NO: 72. In some embodiments, the protein isidentical to SEQ ID NO:72 except for the presence of an amino acidmutation comprising any one mutation selected from the group consistingof: N60A, R62A, R80A, L88A, P93A, P99A, and P115A or selected from thegroup consisting of: N60A, R62A, R80A, L88A, and P93A, numberedaccording to SEQ ID NO: 72. In some embodiments, the protein isidentical to SEQ ID NO:72 except for the presence of any two, three,four, five, six, or seven mutations selected from the group consistingof: W58A, N60A, R62A, R80A, L88A, P93A, P99A, and P115A or selected fromthe group consisting of N60A, R62A, R80A, L88A, and P93A, numberedaccording to SEQ ID NO: 72. In some embodiments, the protein isidentical to SEQ ID NO:72 except for the presence of the amino acidsubstitutions W58A, N60A, R62A, R80A, L88A, P93A, P99A, and P115A orexcept for the presence of the amino acid substitutions N60A, R62A,R80A, L88A, and P93A, numbered according to SEQ ID NO: 72.

In certain embodiments, the epitope of an antigen-binding domaindescribed herein is used as an immunogen to produce antibodies. See,e.g., Section 7.3 infra for methods for producing antibodies.

In specific aspects, an antigen-binding domain described herein, whichimmunospecifically binds to OX40 (e.g., human OX40), functions as anagonist when present in a monospecific bivalent format.

In certain embodiments, an antigen-binding domain described herein,which immunospecifically binds to OX40 (e.g., human OX40), increasesOX40 (e.g., human OX40) activity, when present in a monospecificbivalent form, by at least about 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold,2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold as assessed bymethods described herein and/or known to one of skill in the art,relative to OX40 (e.g., human OX40) activity without any antibody orwith an unrelated antibody (e.g., an antibody that does notimmunospecifically bind to OX40). In certain embodiments, anantigen-binding domain described herein, which immunospecifically bindsto OX40 (e.g., human OX40), increases OX40 (e.g., human OX40) activity,when present in a monospecific bivalent form, by at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98%, or 99% as assessed by methods described herein and/orknown to one of skill in the art, relative to OX40 (e.g., human OX40)activity without any antibody or with an unrelated antibody (e.g., anantibody that does not immunospecifically bind to OX40). Non-limitingexamples of OX40 (e.g., human OX40) activity can include OX40 (e.g.,human OX40) signaling, cell proliferation, cell survival, and cytokineproduction (e.g., IL-2, TNF-α, IFN-γ, IL-4, IL-10, and/or IL-13). Incertain embodiments, an antigen-binding domain described herein, whichimmunospecifically binds to OX40 (e.g., human OX40), induces, enhances,or increases an OX40 (e.g., human OX40) activity, when present in amonospecific bivalent form. In specific embodiments, an increase in anOX40 activity is assessed as described in the Examples, infra.

In certain embodiments, a multispecific (e.g., bispecific) antibodyprovided herein comprises an antigen-binding domain that binds to OX40as described in U.S. Application No. 62/161,198, which is hereinincorporated by reference in its entirety. 7.2.2 GITR Antigen-BindingDomains

In a particular embodiment, an antigen-binding domain described herein,which specifically binds to GITR (e.g., human GITR), comprises a lightchain variable region (VL) comprising:

-   -   (a) a VL-CDR1 comprising the amino acid sequence of        KSSQSLLNSX₁NQKNYLX₂ (SEQ ID NO:90), wherein X₁ is G or S; and X₂        is T or S;    -   (b) a VL-CDR2 comprising the amino acid sequence of WASTRES (SEQ        ID NO:5); and    -   (c) a VL-CDR3 comprising the amino acid sequence of QNX₁YSX₂PYT        (SEQ ID NO:92), wherein X₁ is D or E; and X₂ is Y, F or S, as        shown in Table 7.

In another embodiment, a GITR antigen-binding domain described herein,which specifically binds to GITR (e.g., human GITR), comprises acomprising a heavy chain variable region (VH) comprising:

-   -   (a) a VH-CDR1 comprising the amino acid sequence of X₁YX₂MX₃(SEQ        ID NO:87), wherein X₁ is D, E or G; X₂ is A or V; and X₃ is Y or        H;    -   (b) a VH-CDR2 comprising the amino acid sequence of        X₁IX₂TX₃SGX₄X₅X₆YNQKFX₇X₈(SEQ ID NO:88), wherein X₁ is V or L;        X₂ is R, K or Q; X₃ is Y or F; X₄ is D, E or G; X₅ is V or L; X₆        is T or S; X₇ is K, R or Q; and X₈ is D, E or G;    -   (c) a VH-CDR3 comprising the amino acid sequence of SGTVRGFAY        (SEQ ID NO:3), as shown in Table 8.

In another particular embodiment, an antigen-binding domain describedherein, which specifically binds to GITR (e.g., human GITR), comprises alight chain variable region (VL) comprising:

-   -   (a) a VL-CDR1 comprising the amino acid sequence of        KSSQSLLNSX₁NQKNYLT (SEQ ID NO:4), wherein X₁ is G or S;    -   (b) a VL-CDR2 comprising the amino acid sequence of WASTRES (SEQ        ID NO:5); and    -   (c) a VL-CDR3 comprising the amino acid sequence of QNX₁YSX₂PYT        (SEQ ID NO:6), wherein X₁ is D or E; and X₂ is Y or F, as shown        in Table 7.

In another embodiment, a GITR antigen-binding domain described herein,which specifically binds to GITR (e.g., human GITR), comprises acomprising a heavy chain variable region (VH) comprising:

-   -   (a) a VH-CDR1 comprising the amino acid sequence of X₁YAMX₂ (SEQ        ID NO:1), wherein X₁ is D, G, or E; and X₂ is Y or H;    -   (b) a VH-CDR2 comprising the amino acid sequence of        X₁IRTYSGX₂VX₃YNQKFX₄X₅ (SEQ ID NO:2), wherein X₁ is V or L; X₂        is D or G; X₃ is T or S; X₄ is K, R, or Q; and X₅ is D, E, or G;    -   (c) a VH-CDR3 comprising the amino acid sequence of SGTVRGFAY        (SEQ ID NO:3); as shown in Table 8.

TABLE 7 GITR VL CDR amino acid sequences * VL CDR2 VL CDR3 AntibodyVL CDR1 (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) Consensus 1KSSQSLLNSX₁NQKNYLX₂, WASTRES (5) QNX₁YSX₂PYT,wherein X₁ is G or S; and X₂ is wherein X₁ is D or E; T or S (90)and X₂ is Y, F or S (92) Consensus 2 KSSQSLLNSX₁NQKNYLT WASTRES (5)QNX₁YSX₂PYT X₁ is G or S (4) X₁ is D or E; and X₂ is Y or F (6) pab1876wKSSQSLLNSGNQKNYLT (14) WASTRES (5) QNDYSYPYT (16) pab1967wKSSQSLLNSSNQKNYLT (15) WASTRES (5) QNEYSFPYT (17) pab1975wKSSQSLLNSGNQKNYLT (14) WASTRES (5) QNDYSYPYT (16) pab1979wKSSQSLLNSGNQKNYLT (14) WASTRES (5) QNDYSYPYT (16) * The VL CDRs in Table7 are determined according to Kabat.

TABLE 8 GITR VH CDR amino acid sequences * VH CDR1 VH CDR2 VH CDR3Antibody (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) Consensus 1 X₁YX₂MX₃X₁IX₂TX₃SGX₄X₅X₆YNQKFX₇X₈, SGTVRGFAY (3) wherein X₁ is D,wherein X₁ is V or L; X₂ is R, K or Q; E or G; X₂ is A orX₃ is Y or F; X₄ is D, E or G; X₅ is V or V; and X₃ is Y orL; X₆ is T or S; X₇ is K, R or Q; and X₈ H (87) is D, E or G (88)Consensus 2 X₁YAMX₂ X₁IRTYSGX₂VX₃YNQKFX₄X₅ SGTVRGFAY (3)X₁ is D, G, or E; X₁ is V or L; X₂ is D or G; X₃ is T or S;and X₂ is Y or H X₄ is K, R, or Q; and (1) X₅ is D, E, or G (2) pab1876wDYAMY (7) VIRTYSGDVTYNQKFKD (10) SGTVRGFAY (3) pab1967w GYAMH (8)LIRTYSGGVSYNQKFRE (11) SGTVRGFAY (3) pab1975w EYAMH (9)LIRTYSGGVSYNQKFQG (12) SGTVRGFAY (3) pab1979w EYAMH (9)VIRTYSGGVSYNQKFQE (13) SGTVRGFAY (3) * The VH CDRs in Table 8 aredetermined according to Kabat.

In certain embodiments, provided herein is an antigen-binding domainwhich specifically binds to GITR (e.g., human GITR) and comprises lightchain variable region (VL) CDRs and heavy chain variable region (VH)CDRs of pab1876, pab1967, pab1975, or pab1979, for example as set forthin Tables 1 and 2 (i.e., SEQ ID NOs:14, 5, 16, 7, 10, and 3; SEQ IDNOs:15, 5, 17, 8, 11, and 3; SEQ ID NOs:14, 5, 16, 9, 12, and 3; or SEQID NOs:14, 5, 16, 9, 13, and 3).

In certain embodiments, a GITR antigen-binding domain comprises a lightchain variable framework region that is derived from human IGKV4-1germline sequence (e.g., IGKV4-1*01, e.g., having amino acid sequence ofSEQ ID NO:28).

In certain embodiments, the GITR antigen-binding domain comprises aheavy chain variable framework region that is derived from a humanIGHV1-2 germline sequence (e.g., IGHV1-2*02, e.g., having amino acidsequence of SEQ ID NO:27).

In a specific embodiment, an antigen-binding domain that specificallybinds to GITR (e.g., human GITR) comprises a VL domain comprising theamino acid sequence of SEQ ID NO:19, 21, 23, or 26. In a specificembodiment, an antigen-binding domain that specifically binds to GITR(e.g., human GITR) comprises a VL domain consisting of or consistingessentially of the amino acid sequence of SEQ ID NO:19, 21, 23, or 26.

In certain embodiments, an antigen-binding domain that specificallybinds to GITR (e.g., human GITR) comprises a VH domain comprising theamino acid sequence of SEQ ID NO:18, 20, 22, 24, or 25. In someembodiments, an antigen-binding domain that specifically binds to GITR(e.g., human GITR) comprises a VH domain consisting of or consistingessentially of the amino acid sequence of SEQ ID NO:18, 20, 22, 24, or25.

In certain embodiments, an antigen-binding domain that specificallybinds to GITR (e.g., human GITR) comprises a VH domain and a VL domain,wherein the VH domain and the VL domain comprise the amino acidsequences of SEQ ID NOs:18 and 19; SEQ ID NOs:20 and 21; SEQ ID NOs:22and 23; SEQ ID NOs:24 and 23; or SEQ ID NOs:25 and 26; respectively. Incertain embodiments, an antigen-binding domain that specifically bindsto GITR (e.g., human GITR) comprises a VH domain and a VL domain,wherein the VH domain and the VL domain consist of or consistessentially of the amino acid sequences of SEQ ID NOs:18 and 19; SEQ IDNOs:20 and 21; SEQ ID NOs:22 and 23; SEQ ID NOs:24 and 23; or SEQ IDNOs:25 and 26; respectively, e.g., as shown in Table 9.

TABLE 9 VH and VL sequences of exemplary anti-GITR antibodies AntibodyVH (SEQ ID NO:) VL (SEQ ID NO:) pab1876w 18 19 pab1967w 20 21 pab1975w22 23 pab1979w 24 23

In specific aspects, provided herein is an antigen-binding domaincomprising a light chain and heavy chain, e.g., a separate light chainand heavy chain. With respect to the light chain, in a specificembodiment, the light chain of an antigen-binding domain describedherein is a kappa light chain. In another specific embodiment, the lightchain of an antigen-binding domain described herein is a lambda lightchain. In yet another specific embodiment, the light chain of anantigen-binding domain described herein is a human kappa light chain ora human lambda light chain. In a particular embodiment, anantigen-binding domain described herein, which immunospecifically bindsto a GITR polypeptide (e.g., human GITR) comprises a light chain whereinthe amino acid sequence of the VL domain comprises the sequence setforth in SEQ ID NO:19, 21, 23, or 26 and wherein the constant region ofthe light chain comprises the amino acid sequence of a human kappa lightchain constant region. In another particular embodiment, anantigen-binding domain described herein, which immunospecifically bindsto GITR (e.g., human GITR) comprises a light chain wherein the aminoacid sequence of the VL domain comprises the sequence set forth in SEQID NO:19, 21, 23, or 26 and wherein the constant region of the lightchain comprises the amino acid sequence of a human lambda light chainconstant region. In a specific embodiment, an antigen-binding domaindescribed herein, which immunospecifically binds to GITR (e.g., humanGITR) comprises a light chain wherein the amino acid sequence of the VLdomain comprises the sequence set forth in SEQ ID NO:19, 21, 23, or 26and wherein the constant region of the light chain comprises the aminoacid sequence of a human kappa or lambda light chain constant region.Non-limiting examples of human constant region sequences have beendescribed in the art, e.g., see U.S. Pat. No. 5,693,780 and Kabat E A etal., (1991) supra.

In a particular embodiment, an antigen-binding domain described herein,which specifically binds to GITR (e.g., human GITR) comprises a lightchain comprising the amino acid sequence set forth in SEQ ID NO:37 or38.

With respect to the heavy chain, in a specific embodiment, the heavychain of an antigen-binding domain described herein can be an alpha (α),delta (δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In anotherspecific embodiment, the heavy chain of an antigen-binding domaindescribed can comprise a human alpha (α), delta (δ), epsilon (ε), gamma(γ) or mu (μ) heavy chain. In a particular embodiment, anantigen-binding domain described herein, which immunospecifically bindsto GITR (e.g., human GITR), comprises a heavy chain wherein the aminoacid sequence of the VH domain can comprise the sequence set forth inSEQ ID NO:18, 20, 22, 24, or 25 and wherein the constant region of theheavy chain comprises the amino acid sequence of a human gamma (γ) heavychain constant region. In a specific embodiment, an antigen-bindingdomain described herein, which specifically binds to GITR (e.g., humanGITR), comprises a heavy chain wherein the amino acid sequence of the VHdomain comprises the sequence set forth in SEQ ID NO: 18, 20, 22, 24, or25, and wherein the constant region of the heavy chain comprises theamino acid of a human heavy chain described herein or known in the art.Non-limiting examples of human constant region sequences have beendescribed in the art, e.g., see U.S. Pat. No. 5,693,780 and Kabat E A etal., (1991) supra.

In a particular embodiment, an antigen-binding domain described herein,which specifically binds to GITR (e.g., human GITR), comprises a heavychain comprising the amino acid sequence set forth in SEQ ID NO:31. Inanother embodiment, an antigen-binding domain described herein, whichspecifically binds to GITR (e.g., human GITR), comprises a heavy chaincomprising the amino acid sequence set forth in SEQ ID NO:32. In anotherembodiment, an antigen-binding domain described herein, whichspecifically binds to GITR (e.g., human GITR), comprises a heavy chaincomprising the amino acid sequence set forth in SEQ ID NO:33. In anotherembodiment, an antigen-binding domain described herein, whichspecifically binds to GITR (e.g., human GITR), comprises a heavy chaincomprising the amino acid sequence set forth in SEQ ID NO:34. In anotherembodiment, an antigen-binding domain described herein, whichspecifically binds to GITR (e.g., human GITR), comprises a heavy chaincomprising the amino acid sequence set forth in SEQ ID NO:35. In anotherembodiment, an antigen-binding domain described herein, whichspecifically binds to GITR (e.g., human GITR), comprises a heavy chaincomprising the amino acid sequence set forth in SEQ ID NO:39.

In a specific embodiment, an antigen-binding domain described herein,which immunospecifically binds to GITR (e.g., human GITR) comprises a VLdomain and a VH domain comprising any amino acid sequences describedherein, wherein the constant regions comprise the amino acid sequencesof the constant regions of an IgG, IgE, IgM, IgD, IgA, or IgYimmunoglobulin molecule, or a human IgG, IgE, IgM, IgD, IgA, or IgYimmunoglobulin molecule. In another specific embodiment, anantigen-binding domain described herein, which immunospecifically bindsto GITR (e.g., human GITR) comprises a VL domain and a VH domaincomprising any amino acid sequences described herein, wherein theconstant regions comprise the amino acid sequences of the constantregions of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule,any class (e.g., IgG₁,IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂), or any subclass(e.g., IgG₂a and IgG_(2b)) of immunoglobulin molecule. In a particularembodiment, the constant regions comprise the amino acid sequences ofthe constant regions of a human IgG, IgE, IgM, IgD, IgA, or IgYimmunoglobulin molecule, any class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁,and IgA₂), or any subclass (e.g., IgG_(2a) and IgG_(2b)) ofimmunoglobulin molecule.

In another specific embodiment, an antigen-binding domain describedherein, which immunospecifically binds to GITR (e.g., human GITR),comprises a VL domain and a VH domain comprising any amino acidsequences described herein, wherein the constant regions comprise theamino acid sequences of the constant regions of a human IgG₁ (e.g.,allotypes G1m3, G1m17,1 or Glm17,1,2), human IgG₂, or human IgG₄. In aparticular embodiment, an antigen-binding domain described herein, whichimmunospecifically binds to GITR (e.g., human GITR), comprises a VLdomain and a VH domain comprising any amino acid sequences describedherein, wherein the constant regions comprise the amino acid sequencesof the constant region of a human IgG₁ (allotype Glm3). Non-limitingexamples of human constant regions are described in the art, e.g., seeKabat E A et al., (1991) supra.

In another embodiment, an antigen-binding domain described herein, whichspecifically binds to GITR (e.g., human GITR), comprises a light chaincomprising the amino acid sequence set forth in SEQ ID NO:37 and a heavychain comprising the amino acid sequence set forth in SEQ ID NO:31, 32,33, 34, 35, or 39.

In certain embodiments, an antigen-binding domain described herein,which immunospecifically binds to GITR (e.g., human GITR), comprises aVL domain having at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, or at least 98% sequence identity to theamino acid sequence of the VL domain of pab1876w, pab1967w, pab1975w, orpab1979w (i.e., SEQ ID NO:19, 21, or 23), e.g., wherein theantigen-binding domain comprises VL CDRs that are identical to the VLCDRs of pab1876w, pab1967w, pab1975w, or pab1979w.

In certain embodiments, an antigen-binding domain described herein,which immunospecifically binds to GITR (e.g., human GITR), comprises aVH domain having at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, or at least 98% sequence identity to theamino acid sequence of the VH domain of pab1876w, pab1967w, pab1975w, orpab1979w (i.e., SEQ ID NO:18, 20, 22, or 24), e.g., wherein theantigen-binding domain comprises VH CDRs that are identical to the VHCDRs of pab1876w, pab1967w, pab1975w, or pab1979w.

In certain embodiments, an antigen-binding domain described herein,which immunospecifically binds to GITR (e.g., human GITR), comprises:(i) a VL domain having at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 98% sequence identityto the amino acid sequence of the VL domain of pab1876w, pab1967w,pab1975w, or pab1979w (i.e., SEQ ID NO:19, 21, or 23), and (ii) a VHdomain having at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 98% sequence identity to the aminoacid sequence of the VH domain of pab1876w, pab1967w, pab1975w, orpab1979w (i.e., SEQ ID NO:18, 20, 22, or 24), e.g., wherein the antibodycomprises VL CDRs and VH CDRs that are identical to the VL CDRs and VHCDRs of pab1876w, pab1967w, pab1975w, or pab1979w.

In specific aspects, provided herein is an antigen-binding domain whichcompetes (e.g., in a dose dependent manner) for specific binding to GITR(e.g., human GITR), with an antigen-binding domain comprising a VH andVL domain having the amino acid sequences set forth in SEQ ID NOs:18 and19; SEQ ID NOs:20 and 21, SEQ ID NOs:22 and 23 or SEQ ID NOs:24 and 23,respectively.

In a specific embodiment, an antigen-binding domain described herein isone that is competitively blocked (e.g., in a dose dependent manner) byan antigen-binding domain comprising a VH and VL domain having the aminoacid sequences set forth in SEQ ID NOs:18 and 19; SEQ ID NOs:20 and 21,SEQ ID NOs:22 and 23 or SEQ ID NOs:24 and 23, respectively for specificbinding to GITR (e.g., human GITR).

Assays known to one of skill in the art or described herein (e.g., X-raycrystallography, hydrogen/deuterium exchange coupled with massspectrometry (e.g., liquid chromatography electrospray massspectrometry), alanine scanning, ELISA assays, etc.) can be used todetermine if two antibodies bind to the same epitope.

In a specific embodiment, an antigen-binding domain described hereinimmunospecifically binds to the same epitope as that bound by pab1876w,pab1967w, pab1975w, or pab1979w, or an epitope that overlaps theepitope.

In a specific aspect, the binding between an antigen-binding domaindescribed herein and a variant GITR is substantially weakened relativeto the binding between the antigen-binding domain and a human GITRsequence of residues 26 to 241 of SEQ ID NO: 41, wherein the variantGITR comprises the sequence of residues 26 to 241 of SEQ ID NO: 41except for the presence of a D60A or G63A mutation, numbered accordingto SEQ ID NO: 41. In some embodiments, the variant GITR comprises thesequence of residues 26 to 241 of SEQ ID NO: 41 except for the presenceof a D60A and a G63A mutation, numbered according to SEQ ID NO: 41.

In a specific aspect, an antigen-binding domain described herein bindsto an epitope of a human GITR sequence comprising, consistingessentially of, or consisting of at least one residue in amino acids60-63 of SEQ ID NO:41. In some embodiments, the epitope comprises,consists essentially of, or consists of amino acids 60-63 of SEQ IDNO:41.

In a specific embodiment, an antigen-binding domain described hereinbinds to an epitope of human GITR comprising, consisting essentially of,or consisting of a residue selected from the group consisting of:residues 60, 62, and 63, and a combination thereof of SEQ ID NO:41. Insome embodiments, the epitope comprises, consists essentially of, orconsists of any one residue, or any two, or three residues, selectedfrom the group consisting of: residues 60, 62, and 63 of SEQ ID NO:41.

In a specific aspect, an antigen-binding domain described hereinexhibits, as compared to binding to a human GITR sequence of residues 26to 241 of SEQ ID NO: 41, reduced or absent binding to a proteinidentical to residues 26 to 241 of SEQ ID NO: 41 except for the presenceof an amino acid mutation (e.g., substitution) selected from the groupconsisting of: D60A and G63A, numbered according to SEQ ID NO: 41. Insome embodiments, the substitution is D60A, numbered according to SEQ IDNO: 41. In some embodiments, the substitution is G63A, numberedaccording to SEQ ID NO: 41.

In certain embodiments, the epitope of an antigen-binding domaindescribed herein is used as an immunogen to produce antibodies. See,e.g., Section 7.3 infra for methods for producing antibodies.

In specific aspects, an antigen-binding domain described herein, whichimmunospecifically binds to GITR (e.g., human GITR), functions as anagonist when present in a monospecific bivalent format.

In certain embodiments, an antigen-binding domain described herein,which immunospecifically binds to GITR (e.g., human GITR), increasesGITR (e.g., human GITR) activity, when present in a monospecificbivalent form, by at least about 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold,2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold as assessed bymethods described herein and/or known to one of skill in the art,relative to GITR (e.g., human GITR) activity without any antibody orwith an unrelated antibody (e.g., an antibody that does notimmunospecifically bind to GITR). In certain embodiments, anantigen-binding domain described herein, which immunospecifically bindsto GITR (e.g., human GITR), increases GITR (e.g., human GITR) activity,when present in a monospecific bivalent form, by at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98%, or 99% as assessed by methods described herein and/orknown to one of skill in the art, relative to GITR (e.g., human GITR)activity without any antibody or with an unrelated antibody (e.g., anantibody that does not immunospecifically bind to GITR). Non-limitingexamples of GITR (e.g., human GITR) activity can include GITR (e.g.,human GITR) signaling, cell proliferation, cell survival, and cytokineproduction (e.g., IL-2, TNF-α, IFN-γ, IL-4, IL-10, and/or IL-13). Incertain embodiments, an antigen-binding domain described herein, whichimmunospecifically binds to GITR (e.g., human GITR), induces, enhances,or increases a GITR (e.g., human GITR) activity, when present in amonospecific bivalent form. In specific embodiments, an increase in aGITR activity is assessed as described in the Examples, infra.

In certain embodiments, a multispecific (e.g., bispecific) antibodyprovided herein comprises an antigen-binding domain that binds to GITRas described in International Application No. PCT/US2015/032895, whichis herein incorporated by reference in its entirety.

7.2.3 Antigen-Binding Domains

In certain aspects, an antigen-binding domain described herein may bedescribed by its VL domain alone, or its VH domain alone, or by its 3 VLCDRs alone, or its 3 VH CDRs alone. See, for example, Rader C et al.,(1998) PNAS 95: 8910-8915, which is incorporated herein by reference inits entirety, describing the humanization of the mouse anti-αvβ3antibody by identifying a complementing light chain or heavy chain,respectively, from a human light chain or heavy chain library, resultingin humanized antibody variants having affinities as high or higher thanthe affinity of the original antibody. See also Clackson T et al.,(1991) Nature 352: 624-628, which is incorporated herein by reference inits entirety, describing methods of producing antibodies that bind aspecific antigen by using a specific VL domain (or VH domain) andscreening a library for the complementary variable domains. The screenproduced 14 new partners for a specific VH domain and 13 new partnersfor a specific VL domain, which were strong binders, as determined byELISA. See also Kim S J & Hong H J, (2007) J Microbiol 45: 572-577,which is incorporated herein by reference in its entirety, describingmethods of producing antibodies that bind a specific antigen by using aspecific VH domain and screening a library (e.g., human VL library) forcomplementary VL domains; the selected VL domains in turn could be usedto guide selection of additional complementary (e.g., human) VH domains.

In certain aspects, the CDRs of an antigen-binding domain can bedetermined according to the Chothia numbering scheme, which refers tothe location of immunoglobulin structural loops (see, e.g., Chothia C &Lesk A M, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997)J Mol Biol 273: 927-948; Chothia C et al., (1992) J Mol Biol 227:799-817; Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and U.S.Pat. No. 7,709,226). Typically, when using the Kabat numberingconvention, the Chothia CDR-H1 loop is present at heavy chain aminoacids 26 to 32, 33, or 34, the Chothia CDR-H2 loop is present at heavychain amino acids 52 to 56, and the Chothia CDR-H3 loop is present atheavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop ispresent at light chain amino acids 24 to 34, the Chothia CDR-L2 loop ispresent at light chain amino acids 50 to 56, and the Chothia CDR-L3 loopis present at light chain amino acids 89 to 97. The end of the ChothiaCDR-H1 loop when numbered using the Kabat numbering convention variesbetween H32 and H34 depending on the length of the loop (this is becausethe Kabat numbering scheme places the insertions at H35A and H35B; ifneither 35A nor 35B is present, the loop ends at 32; if only 35A ispresent, the loop ends at 33; if both 35A and 35B are present, the loopends at 34).

In certain aspects, provided herein are antigen-binding domains thatspecifically bind to GITR or OX40 (e.g., human GITR or OX40) andcomprise the Chothia VL CDRs of a VL of pab1876w, pab1967w, pab1975w,pab1979w, pab2049w, or pab1949w. In certain aspects, provided herein areantigen-binding domains that specifically bind to GITR or OX40 (e.g.,human GITR or OX40) and comprise the Chothia VH CDRs of a VH of pab1876,pab1967, pab1975, pab1979, pab2049, or pab1949. In certain aspects,provided herein are antigen-binding domains that specifically bind toGITR or OX40 (e.g., human GITR or OX40) and comprise the Chothia VL CDRsof a VL of pab1876, pab1967, pab1975w, pab1979w, pab2049w, or pab1949wand comprise the Chothia VH CDRs of a VH of pab1876w, pab1967w,pab1975w, pab1979w, pab2049w, or pab1949w. In certain embodiments,antigen-binding domains that specifically bind to GITR or OX40 (e.g.,human GITR or OX40) comprise one or more CDRs, in which the Chothia andKabat CDRs have the same amino acid sequence. In certain embodiments,provided herein are antigen-binding domains that specifically bind toGITR or OX40 (e.g., human GITR or OX40) and comprise combinations ofKabat CDRs and Chothia CDRs.

In certain aspects, the CDRs of an antigen-binding domain can bedetermined according to the IMGT numbering system as described inLefranc M-P, (1999) The Immunologist 7: 132-136 and Lefranc M-P et al.,(1999) Nucleic Acids Res 27: 209-212. According to the IMGT numberingscheme, VH-CDR1 is at positions 26 to 35, VH-CDR2 is at positions 51 to57, VH-CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to 32,VL-CDR2 is at positions 50 to 52, and VL-CDR3 is at positions 89 to 97.In a particular embodiment, provided herein are antigen-binding domainsthat specifically bind to GITR or OX40 (e.g., human GITR or OX40) andcomprise CDRs of pab1876w, pab1967w, pab1975w, pab1979w, pab2049w, orpab1949w as determined by the IMGT numbering system, for example, asdescribed in Lefranc M-P (1999) supra and Lefranc M-P et al., (1999)supra).

In certain aspects, the CDRs of an antigen-binding domain can bedetermined according to MacCallum R M et al., (1996) J Mol Biol 262:732-745. See also, e.g., Martin A. “Protein Sequence and StructureAnalysis of Antibody Variable Domains,” in Antibody Engineering,Kontermann and Dilbel, eds., Chapter 31, pp. 422-439, Springer-Verlag,Berlin (2001). In a particular embodiment, provided herein areantigen-binding domains that specifically bind to GITR or OX40 (e.g.,human GITR or OX40) and comprise CDRs of pab1876w, pab1967w, pab1975w,pab1979w, pab2049w, or pab1949w as determined by the method in MacCallumR M et al.

In certain aspects, the CDRs of an antibody can be determined accordingto the AbM numbering scheme, which refers AbM hypervariable regionswhich represent a compromise between the Kabat CDRs and Chothiastructural loops, and are used by Oxford Molecular's AbM antibodymodeling software (Oxford Molecular Group, Inc.). In a particularembodiment, provided herein are antigen-binding domains thatspecifically bind to GITR or OX40 (e.g., human GITR or OX40) andcomprise CDRs of pab1876w, pab1967w, pab1975w, pab1979w, pab2049w, orpab1949w as determined by the AbM numbering scheme.

In a specific embodiment, the position of one or more CDRs along the VH(e.g., CDR1, CDR2, or CDR3) and/or VL (e.g., CDR1, CDR2, or CDR3) regionof an antigen-binding domain described herein may vary by one, two,three, four, five, or six amino acid positions so long as immunospecificbinding to GITR or OX40 (e.g., human GITR or OX40) is maintained (e.g.,substantially maintained, for example, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%). For example, inone embodiment, the position defining a CDR of an antigen-binding domaindescribed herein can vary by shifting the N-terminal and/or C-terminalboundary of the CDR by one, two, three, four, five, or six amino acids,relative to the CDR position of an antigen-binding domain describedherein, so long as immunospecific binding to GITR or OX40 (e.g., humanGITR or OX40) is maintained (e.g., substantially maintained, forexample, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%). In another embodiment, the length of one ormore CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and/or VL (e.g.,CDR1, CDR2, or CDR3) region of an antigen-binding domain describedherein may vary (e.g., be shorter or longer) by one, two, three, four,five, or more amino acids, so long as immunospecific binding to GITR orOX40 (e.g., human GITR or OX40) is maintained (e.g., substantiallymaintained, for example, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%).

In one embodiment, a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/orVH CDR3 described herein may be one, two, three, four, five or moreamino acids shorter than one or more of the CDRs described herein (e.g.,SEQ ID NOs:1-6, SEQ ID NOs:87, 88, 3, 90, 5, and 92; SEQ ID NOS:7, 10,3, 14, 5, and 16; SEQ ID NOs:8, 11, 3, 15, 5, and 17; SEQ ID NOs:9, 12,3, 14, 5, and 16; SEQ ID NOs:9, 13, 3, 14, 5, and 16; SEQ ID NOs:47-52,or SEQ ID NOs:47-51 and 53) so long as immunospecific binding to GITR orOX40 (e.g., human GITR or OX40) is maintained (e.g., substantiallymaintained, for example, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%). In another embodiment, a VLCDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH CDR3 describedherein may be one, two, three, four, five or more amino acids longerthan one or more of the CDRs described herein (e.g., SEQ ID NOs:1-6, SEQID NOs:87, 88, 3, 90, 5, and 92; SEQ ID NOS:7, 10, 3, 14, 5, and 16; SEQID NOs:8, 11, 3, 15, 5, and 17; SEQ ID NOs:9, 12, 3, 14, 5, and 16; SEQID NOs:9, 13, 3, 14, 5, and 16; SEQ ID NOs:47-52, or SEQ ID NOs:47-51and 53) so long as immunospecific binding to GITR or OX40 (e.g., humanGITR or OX40) is maintained (e.g., substantially maintained, forexample, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%). In another embodiment, the amino terminus of aVL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH CDR3 describedherein may be extended by one, two, three, four, five or more aminoacids compared to one or more of the CDRs described herein (e.g., SEQ IDNOs:1-6, SEQ ID NOs:87, 88, 3, 90, 5, and 92; SEQ ID NOS:7, 10, 3, 14,5, and 16; SEQ ID NOs:8, 11, 3, 15, and 17; SEQ ID NOs:9, 12, 3, 14, 5,and 16; SEQ ID NOs:9, 13, 3, 14, 5, and 16; SEQ ID NOs:47-52, or SEQ IDNOs:47-51 and 53) so long as immunospecific binding to GITR or OX40(e.g., human GITR OR OX40) is maintained (e.g., substantiallymaintained, for example, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%). In another embodiment, thecarboxy terminus of a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2,and/or VH CDR3 described herein may be extended by one, two, three,four, five or more amino acids compared to one or more of the CDRsdescribed herein (e.g., SEQ ID NO:1-6) so long as immunospecific bindingto GITR OR OX40 (e.g., human GITR OR OX40) is maintained (e.g.,substantially maintained, for example, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%). In anotherembodiment, the amino terminus of a VL CDR1, VL CDR2, VL CDR3, VH CDR1,VH CDR2, and/or VH CDR3 described herein may be shortened by one, two,three, four, five or more amino acids compared to one or more of theCDRs described herein (e.g., SEQ ID NO:1-6) so long as immunospecificbinding to GITR OR OX40 (e.g., human GITR OR OX40) is maintained (e.g.,substantially maintained, for example, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%). In one embodiment,the carboxy terminus of a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2,and/or VH CDR3 described herein may be shortened by one, two, three,four, five or more amino acids compared to one or more of the CDRsdescribed herein (e.g., SEQ ID NOs:1-6, SEQ ID NOs:87, 88, 3, 90, 5, and92; SEQ ID NOS:7, 10, 3, 14, 5, and 16; SEQ ID NOs:8, 11, 3, 15, 5, and17; SEQ ID NOs:9, 12, 3, 14, 5, and 16; SEQ ID NOs:9, 13, 3, 14, 5, and16; SEQ ID NOs:47-52, or SEQ ID NOs:47-51 and 53) so long asimmunospecific binding to GITR OR OX40 (e.g., human GITR OR OX40) ismaintained (e.g., substantially maintained, for example, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).Any method known in the art can be used to ascertain whetherimmunospecific binding to GITR OR OX40 (e.g., human GITR OR OX40) ismaintained, for example, the binding assays and conditions described inthe “Examples” section (Section 8) provided herein.

In another particular embodiment, an antigen-binding domain describedherein, which immunospecifically binds to GITR or OX40 (e.g., human GITRor OX40), comprises a heavy chain and a light chain, wherein (i) theheavy and light chains comprise a VH domain and a VL domain,respectively, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2,and VL CDR3 of the VH and VL domains comprise the amino acid sequencesset forth in SEQ ID NOs:1-6, SEQ ID NOs:87, 88, 3, 90, 5, and 92; SEQ IDNOS:7, 10, 3, 14, 5, and 16; SEQ ID NOs:8, 11, 3, 15, 5, and 17; SEQ IDNOs:9, 12, 3, 14, 5, and 16; SEQ ID NOs:9, 13, 3, 14, 5, and 16; SEQ IDNOs:47-52, or SEQ ID NOs:47-51 and 53, respectively; (ii) the lightchain further comprises a constant light chain domain comprising theamino acid sequence of the constant domain of a human kappa light chain;and (iii) the heavy chain further comprises a constant heavy chaindomain comprising the amino acid sequence of the constant domain of ahuman IgG₁ (optionally Ig (allotype Glm3)) heavy chain.

In another particular embodiment, an antigen-binding domain describedherein, which immunospecifically binds to GITR or OX40 (e.g., human GITRor OX40), comprises a heavy chain and a light chain, wherein (i) theheavy and light chains comprise a VH domain and a VL domain,respectively comprising the amino acid sequences set forth in SEQ IDNOs:18 and 19, SEQ ID NOs:20 and 21, SEQ ID NOs:22 and 23, SEQ ID NOs:24and 23, SEQ ID NOs:25 and 26, SEQ ID NOs:54 and 55, or SEQ ID NOs:54 and56, respectively; (ii) the light chain further comprises a constantdomain comprising the amino acid sequence of the constant domain of ahuman kappa light chain; and (iii) the heavy chain further comprises aconstant domain comprising the amino acid sequence of the constantdomain of a human IgG₁ (optionally IgG₁ (allotype Glm3)) heavy chain.

In another particular embodiment, an antigen-binding domain describedherein, which immunospecifically binds to GITR or OX40 (e.g., human GITRor OX40), comprises a heavy chain and a light chain, wherein (i) theheavy and light chains comprise a VH domain and a VL domain,respectively, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2,and VL CDR3 of the VH and VL domains comprise the amino acid sequencesset forth in SEQ ID NOs:1-6, SEQ ID NOs:87, 88, 3, 90, 5, and 92; SEQ IDNOS:7, 10, 3, 14, 5, and 16; SEQ ID NOs:8, 11, 3, 15, 5, and 17; SEQ IDNOs:9, 12, 3, 14, 5, and 16; SEQ ID NOs:9, 13, 3, 14, 5, and 16; SEQ IDNOs:47-52, or SEQ ID NOs:47-51 and 53, respectively; (ii) the lightchain further comprises a constant light chain domain comprising theamino acid sequence of the constant domain of a human kappa light chain;and (iii) the heavy chain further comprises a constant heavy chaindomain comprising the amino acid sequence of the constant domain of ahuman IgG₄ heavy chain.

In another particular embodiment, an antigen-binding domain describedherein, which immunospecifically binds to GITR or OX40 (e.g., human GITRor OX40), comprises a heavy chain and a light chain, wherein (i) theheavy and light chains comprise a VH domain and a VL domain,respectively comprising the amino acid sequences set forth in SEQ IDNOs:18 and 19, SEQ ID NOs:20 and 21, SEQ ID NOs:22 and 23, SEQ ID NOs:24and 23, SEQ ID NOs:25 and 26, SEQ ID NOs:54 and 55, or SEQ ID NOs:54 and56, respectively; (ii) the light chain further comprises a constantdomain comprising the amino acid sequence of the constant domain of ahuman kappa light chain; and (iii) the heavy chain further comprises aconstant domain comprising the amino acid sequence of the constantdomain of a human IgG₄ heavy chain.

In another particular embodiment, an antigen-binding domain describedherein, which immunospecifically binds to GITR or OX40 (e.g., human GITRor OX40), comprises a heavy chain and a light chain, wherein (i) theheavy and light chains comprise a VH domain and a VL domain,respectively, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2,and VL CDR3 of the VH and VL domains comprise the amino acid sequencesset forth in SEQ ID NOs:1-6, SEQ ID NOs:87, 88, 3, 90, 5, and 92; SEQ IDNOS:7, 10, 3, 14, 5, and 16; SEQ ID NOs:8, 11, 3, 15, 5, and 17; SEQ IDNOs:9, 12, 3, 14, 5, and 16; SEQ ID NOs:9, 13, 3, 14, 5, and 16; SEQ IDNOs:47-52, or SEQ ID NOs:47-51 and 53, respectively; (ii) the lightchain further comprises a constant light chain domain comprising theamino acid sequence of the constant domain of a human kappa light chain;and (iii) the heavy chain further comprises a constant heavy chaindomain comprising the amino acid sequence of the constant domain of ahuman IgG₂ heavy chain.

In another particular embodiment, an antigen-binding domain describedherein, which immunospecifically binds to GITR or OX40 (e.g., human GITRor OX40), comprises a heavy chain and a light chain, wherein (i) theheavy and light chains comprise a VH domain and a VL domain,respectively comprising the amino acid sequences set forth in SEQ IDNOs:18 and 19, SEQ ID NOs:20 and 21, SEQ ID NOs:22 and 23, SEQ ID NOs:24and 23, SEQ ID NOs:25 and 26, SEQ ID NOs:54 and 55, or SEQ ID NOs:54 and56, respectively; (ii) the light chain further comprises a constantdomain comprising the amino acid sequence of the constant domain of ahuman kappa light chain; and (iii) the heavy chain further comprises aconstant domain comprising the amino acid sequence of the constantdomain of a human IgG₂ heavy chain.

In another particular embodiment, an antigen-binding domain describedherein, which immunospecifically binds to GITR or OX40 (e.g., human GITRor OX40), comprises a heavy chain and a light chain, wherein (a) a heavychain comprising the amino acid sequence of SEQ ID NO: 59 with an aminoacid substitution of N to A or Q at amino acid position 297; and (b) alight chain comprising the amino acid sequence of SEQ ID NO: 67 or 69.

In another particular embodiment, an antigen-binding domain describedherein, which immunospecifically binds to GITR or OX40 (e.g., human GITRor OX40), comprises (a) a heavy chain comprising the amino acid sequenceof SEQ ID NO: 59 or 29 with an amino acid substitution selected from thegroup consisting of: S to E at amino acid position 267, L to F at aminoacid position 328, and both S to E at amino acid position 267 and L to Fat amino acid position 328; and (b) a light chain comprising the aminoacid sequence of SEQ ID NO: 67 or 69, or SEQ ID NO:37.

In specific embodiments, an antigen-binding domains described herein,which immunospecifically binds to GITR or OX40 (e.g., human GITR orOX40), comprises framework regions (e.g., framework regions of the VLdomain and/or VH domain) that are human framework regions or derivedfrom human framework regions. Non-limiting examples of human frameworkregions are described in the art, e.g., see Kabat E A et al., (1991)supra). In certain embodiment, an antigen-binding domain describedherein comprises framework regions (e.g., framework regions of the VLdomain and/or VH domain) that are primate (e.g., non-human primate)framework regions or derived from primate (e.g., non-human primate)framework regions.

For example, CDRs from antigen-specific non-human antibodies, typicallyof rodent origin (e.g., mouse or rat), are grafted onto homologous humanor non-human primate acceptor frameworks. In one embodiment, thenon-human primate acceptor frameworks are from Old World apes. In aspecific embodiment, the Old World ape acceptor framework is from Pantroglodytes, Pan paniscus or Gorilla gorilla. In a particularembodiment, the non-human primate acceptor frameworks are from thechimpanzee Pan troglodytes. In a particular embodiment, the non-humanprimate acceptor frameworks are Old World monkey acceptor frameworks. Ina specific embodiment, the Old World monkey acceptor frameworks are fromthe genus Macaca. In a certain embodiment, the non-human primateacceptor frameworks are derived from the cynomolgus monkey Macacacynomolgus. Non-human primate framework sequences are described in U.S.Patent Application Publication No. US 2005/0208625.

In another aspect, provided herein are antibodies that containantigen-binding domains that bind the same or an overlapping epitope ofGITR or OX40 (e.g., an epitope of human GITR or OX40) as an antibodydescribed herein (e.g., pab1949w, pab2049w, or pab1876w). In certainembodiments, the epitope of an antibody can be determined by, e.g., NMRspectroscopy, X-ray diffraction crystallography studies, ELISA assays,hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquidchromatography electrospray mass spectrometry), array-basedoligo-peptide scanning assays, and/or mutagenesis mapping (e.g.,site-directed mutagenesis mapping). For X-ray crystallography,crystallization may be accomplished using any of the known methods inthe art (e.g., Giege R et al., (1994) Acta Crystallogr D BiolCrystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen N E (1997) Structure 5: 1269-1274; McPherson A (1976) JBiol Chem 251: 6300-6303). Antibody:antigen crystals may be studiedusing well known X-ray diffraction techniques and may be refined usingcomputer software such as X-PLOR (Yale University, 1992, distributed byMolecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114& 115, eds Wyckoff H W et al.; U.S. Patent Application No.2004/0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D BiolCrystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter C W; Roversi P et al., (2000) Acta Crystallogr D BiolCrystallogr 56(Pt 10): 1316-1323). Mutagenesis mapping studies may beaccomplished using any method known to one of skill in the art. See,e.g., Champe M et al., (1995) supra and Cunningham B C & Wells J A(1989) supra for a description of mutagenesis techniques, includingalanine scanning mutagenesis techniques. In a specific embodiment, theepitope of an antigen-binding fragment is determined using alaninescanning mutagenesis studies. In addition, antigen-binding fragmentsthat recognize and bind to the same or overlapping epitopes of GITRand/or OX40 (e.g., human GITR and/or OX40) can be identified usingroutine techniques such as an immunoassay, for example, by showing theability of one antibody to block the binding of another antibody to atarget antigen, i.e., a competitive binding assay. Competition bindingassays also can be used to determine whether two antibodies have similarbinding specificity for an epitope. Competitive binding can bedetermined in an assay in which the immunoglobulin under test inhibitsspecific binding of a reference antibody to a common antigen, such asGITR or OX40. Numerous types of competitive binding assays are known,for example: solid phase direct or indirect radioimmunoassay (RIA),solid phase direct or indirect enzyme immunoassay (EIA), sandwichcompetition assay (see Stahli C et al., (1983) Methods Enzymol 9:242-253); solid phase direct biotin-avidin EIA (see Kirkland T N et al.,(1986) J Immunol 137: 3614-9); solid phase direct labeled assay, solidphase direct labeled sandwich assay (see Harlow E & Lane D, (1988)Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid phasedirect label RIA using I-125 label (see Morel G A et al., (1988) MolImmunol 25(1): 7-15); solid phase direct biotin-avidin EIA (Cheung R Cet al., (1990) Virology 176: 546-52); and direct labeled RIA.(Moldenhauer G et al., (1990) Scand J Immunol 32: 77-82). Typically,such an assay involves the use of purified antigen (e.g., GITR or OX40such as human GITR or OX40) bound to a solid surface or cells bearingeither of these, an unlabeled test immunoglobulin and a labeledreference immunoglobulin. Competitive inhibition can be measured bydetermining the amount of label bound to the solid surface or cells inthe presence of the test immunoglobulin. Usually the test immunoglobulinis present in excess. Usually, when a competing antibody is present inexcess, it will inhibit specific binding of a reference antibody to acommon antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% ormore. A competition binding assay can be configured in a large number ofdifferent formats using either labeled antigen or labeled antibody. In acommon version of this assay, the antigen is immobilized on a 96-wellplate. The ability of unlabeled antibodies to block the binding oflabeled antibodies to the antigen is then measured using radioactive orenzyme labels. For further details see, for example, Wagener C et al.,(1983) J Immunol 130: 2308-2315; Wagener C et al., (1984) J ImmunolMethods 68: 269-274; Kuroki M et al., (1990) Cancer Res 50: 4872-4879;Kuroki M et al., (1992) Immunol Invest 21: 523-538; Kuroki M et al.,(1992) Hybridoma 11: 391-407 and Antibodies: A Laboratory Manual, EdHarlow E & Lane D editors supra, pp. 386-389.

In one embodiment, a competition assay is performed using surfaceplasmon resonance (BIAcore®), e.g., by an ‘in tandem approach’ such asthat described by Abdiche Y N et al., (2009) Analytical Biochem 386:172-180, whereby GITR or OX40 antigen is immobilized on the chipsurface, for example, a CM5 sensor chip and the anti-GITR or OX40antibodies are then run over the chip. To determine if an antibodycompetes with an anti-GITR or OX40 antigen-binding domain describedherein, the antibody containing the anti-GITR or OX40 antigen-bindingdomain is first run over the chip surface to achieve saturation and thenthe potential, competing antibody is added. Binding of the competingantibody can then be determined and quantified relative to anon-competing control.

In certain aspects, competition binding assays can be used to determinewhether an antibody is competitively blocked, e.g., in a dose dependentmanner, by another antibody for example, an antibody binds essentiallythe same epitope, or overlapping epitopes, as a reference antibody, whenthe two antibodies recognize identical or sterically overlappingepitopes in competition binding assays such as competition ELISA assays,which can be configured in all number of different formats, using eitherlabeled antigen or labeled antibody. In a particular embodiment, anantibody can be tested in competition binding assays with an antibodydescribed herein (e.g., antibody pab1949w, pab2049w, or pab1876w), or achimeric or Fab antibody thereof, or an antibody comprising VH CDRs andVL CDRs of an antibody described herein (e.g., pab1949w, pab2049w, orpab1876w).

In another aspect, provided herein are antigen-binding domains thatcompete (e.g., in a dose dependent manner) for binding to GITR or OX40(e.g., human OX40) with an antigen-binding domain described herein, asdetermined using assays known to one of skill in the art or describedherein (e.g., ELISA competitive assays or surface plasmon resonance). Inanother aspect, provided herein are antigen-binding domains thatcompetitively inhibit (e.g., in a dose dependent manner) anantigen-binding domain described herein (e.g., pab1949w, pab2049w, orpab1876w) from binding to GITR or OX40 (e.g., human GITR or OX40), asdetermined using assays known to one of skill in the art or describedherein (e.g., ELISA competitive assays, or suspension array or surfaceplasmon resonance assay). In specific aspects, provided herein is anantigen-binding fragment which competes (e.g., in a dose dependentmanner) for specific binding to GITR or OX40 (e.g., human GITR or OX40),with an antibody comprising the amino acid sequences described herein(e.g., VL and/or VH amino acid sequences of pab1949w, pab2049w, orpab1876w), as determined using assays known to one of skill in the artor described herein (e.g., ELISA competitive assays, or suspension arrayor surface plasmon resonance assay).

In certain embodiments, provided herein is an antigen-binding fragmentthat competes with an antigen-binding fragment described herein forbinding to GITR or OX40 (e.g., human GITR or OX40) to the same extentthat the antigen-binding fragment described herein self-competes forbinding to GITR or OX40 (e.g., human GITR or OX40). In some embodiments,provided herein is a first antigen-binding antibody fragment thatcompetes with an antigen-binding antibody fragment described herein forbinding to GITR or OX40 (e.g., human GITR or OX40), wherein the firstantigen-binding antibody fragment competes for binding in an assaycomprising the following steps: (a) incubating GITR and/orOX40-transfected cells with the first antigen-binding antibody fragmentin unlabeled form in a container; and (b) adding an antigen-bindingantibody fragment described herein in labeled form in the container andincubating the cells in the container; and (c) detecting the binding ofthe antigen-binding antibody fragment described herein in labeled formto the cells. In certain embodiments, provided herein is a firstantigen-binding antibody fragment that competes with an antigen-bindingantibody fragment described herein for binding to GITR or OX40 (e.g.,human GITR or OX40), wherein the competition is exhibited as reducedbinding of the first antigen-binding antibody fragment to GITR or OX40by more than 80% (e.g., 85%, 90%, 95%, or 98%, or between 80% to 85%,80% to 90%, 85% to 90%, or 85% to 95%).

7.3 Antibody Production

Multi specific (e.g., bispecific) antibodies that immunospecificallybind to GITR and/or OX40 (e.g., human GITR and human OX40) can beproduced by any method known in the art for the synthesis of antibodies,for example, by chemical synthesis or by recombinant expressiontechniques. The methods described herein employ, unless otherwiseindicated, conventional techniques in molecular biology, microbiology,genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR,oligonucleotide synthesis and modification, nucleic acid hybridization,and related fields within the skill of the art. These techniques aredescribed, for example, in the references cited herein and are fullyexplained in the literature. See, e.g., Maniatis T et al., (1982)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress; Sambrook J et al., (1989), Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook Jet al., (2001) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY; Ausubel F M et al.,Current Protocols in Molecular Biology, John Wiley & Sons (1987 andannual updates); Current Protocols in Immunology, John Wiley & Sons(1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: APractical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotidesand Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.)(1999) Genome Analysis: A Laboratory Manual, Cold Spring HarborLaboratory Press.

In a specific embodiment, a multispecific (e.g., bispecific) antibodydescribed herein is a multispecific (e.g., bispecific) antibody (e.g.,recombinant antibody) prepared, expressed, created or isolated by anymeans that involves creation, e.g., via synthesis, genetic engineeringof DNA sequences. In certain embodiments, such a multispecific (e.g.,bispecific) antibody comprises sequences (e.g., DNA sequences or aminoacid sequences) that do not naturally exist within the antibody germlinerepertoire of an animal or mammal (e.g., human) in vivo.

Bispecific, bivalent antibodies, and methods of making them, aredescribed, for instance in U.S. Pat. Nos. 5,731,168, 5,807,706,5,821,333, and U.S. Appl. Publ. Nos. 2003/020734 and 2002/0155537; eachof which is herein incorporated by reference in its entirety. Bispecifictetravalent antibodies, and methods of making them are described, forinstance, in Int. Appl. Publ. Nos. WO02/096948 and WO00/44788, thedisclosures of both of which are herein incorporated by reference in itsentirety. See generally, Int. Appl. Publ. Nos. WO93/17715, WO92/08802,WO91/00360, and WO92/05793; Tutt et al., J. Immunol. 147:60-69 (1991);U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; and5,601,819; and Kostelny et al., J. Immunol. 148:1547-1553 (1992); eachof which is herein incorporated by reference in its entirety.

A bispecific antibody as described herein can be generated according tothe DuoBody technology platform (Genmab A/S) as described, e.g., inInternational Publication Nos. WO 2011/131746, WO 2011/147986, WO2008/119353, and WO 2013/060867, and in Labrijn A F et al., (2013) PNAS110(13): 5145-5150. The DuoBody technology can be used to combine onehalf of a first monospecific antibody containing two heavy and two lightchains with one half of a second monospecific antibody containing twoheavy and two light chains. The resultant heterodimer contains one heavychain and one light chain from the first antibody paired with one heavychain and one light chain from the second antibody. When both of themonospecific antibodies recognize different epitopes on differentantigens, the resultant heterodimer is a bispecific antibody.

The DuoBody technology requires that each of the monospecific antibodiesincludes a heavy chain constant region with a single point mutation inthe CH3 domain. The point mutations allow for a stronger interactionbetween the CH3 domains in the resultant bispecific antibody thanbetween the CH3 domains in either of the monospecific antibodies. Thesingle point mutation in each monospecific antibody is at residue 366,368, 370, 399, 405, 407, or 409, numbered according to the EU numberingsystem, in the CH3 domain of the heavy chain constant region, asdescribed, e.g., in International Publication No. WO 2011/131746.Moreover, the single point mutation is located at a different residue inone monospecific antibody as compared to the other monospecificantibody. For example, one monospecific antibody can comprise themutation F405L (i.e., a mutation from phenylalanine to leucine atresidue 405), while the other monospecific antibody can comprise themutation K409R (i.e., a mutation from lysine to arginine at residue409), numbered according to the EU numbering system. The heavy chainconstant regions of the monospecific antibodies can be an IgG₁, IgG₂,IgG₃, or IgG₄ isotype (e.g., a human IgG₁ isotype), and a bispecificantibody produced by the DuoBody technology can retain Fc-mediatedeffector functions.

Another method for generating bispecific antibodies has been termed the“knobs-into-holes” strategy (see, e.g., Intl. Publ. WO2006/028936). Themispairing of Ig heavy chains is reduced in this technology by mutatingselected amino acids forming the interface of the CH3 domains in IgG. Atpositions within the CH3 domain at which the two heavy chains interactdirectly, an amino acid with a small side chain (hole) is introducedinto the sequence of one heavy chain and an amino acid with a large sidechain (knob) into the counterpart interacting residue location on theother heavy chain. In some embodiments, compositions of the inventionhave immunoglobulin chains in which the CH3 domains have been modifiedby mutating selected amino acids that interact at the interface betweentwo polypeptides so as to preferentially form a bispecific antibody. Thebispecific antibodies can be composed of immunoglobulin chains of thesame subclass (e.g., IgG1 or IgG3) or different subclasses (e.g., IgG1and IgG3, or IgG3 and IgG4).

In one embodiment, a bispecific antibody that binds to GITR and/or OX40comprises a T366W mutation in the “knobs chain” and T366S, L368A, Y407Vmutations in the “hole chain,” and optionally an additional interchaindisulfide bridge between the CH3 domains by, e.g., introducing a Y349Cmutation into the “knobs chain” and a E356C mutation or a S354C mutationinto the “hole chain;” R409D, K370E mutations in the “knobs chain” andD399K, E357K mutations in the “hole chain;” R409D, K370E mutations inthe “knobs chain” and D399K, E357K mutations in the “hole chain;” aT366W mutation in the “knobs chain” and T366S, L368A, Y407V mutations inthe “hole chain;” R409D, K370E mutations in the “knobs chain” and D399K,E357K mutations in the “hole chain;” Y349C, T366W mutations in one ofthe chains and E356C, T366S, L368A, Y407V mutations in the counterpartchain; Y349C, T366W mutations in one chain and S354C, T366S, L368A,Y407V mutations in the counterpart chain; Y349C, T366W mutations in onechain and S354C, T366S, L368A, Y407V mutations in the counterpart chain;and Y349C, T366W mutations in one chain and S354C, T366S, L368A, Y407Vmutations in the counterpart chain (numbering according to the EUnumbering system).

Bispecific antibodies that bind to GITR and/or OX40 can, in someinstances contain, IgG4 and IgG1, IgG4 and IgG2, IgG4 and IgG2, IgG4 andIgG3, or IgG1 and IgG3 chain heterodimers. Such heterodimeric heavychain antibodies, can routinely be engineered by, for example, modifyingselected amino acids forming the interface of the CH3 domains in humanIgG4 and the IgG1 or IgG3 so as to favor heterodimeric heavy chainformation.

In particular embodiments, a multispecific (e.g., bispecific) antibodycan be a chimeric antibody or a humanized antibody. In certainembodiments, a multispecific (e.g., bispecific) antibody can be aF(ab′)₂ fragment. A F(ab′)₂ fragment contains the two antigen-bindingarms of a tetrameric antibody molecule linked by disulfide bonds in thehinge region.

Multispecific (e.g., bispecific) antibodies described herein can begenerated by any technique known to those of skill in the art. Forexample, F(ab′)₂ fragments described herein can be produced byproteolytic cleavage of immunoglobulin molecules, using enzymes such aspepsin.

In a certain aspect, provided herein is a method of making an antibodyor an antigen-binding fragment which immunospecifically binds to GITRand/or OX40 (e.g., human GITR and/or OX40) comprising culturing a cellor cells described herein. In a certain aspect, provided herein is amethod of making an antibody or antigen-binding fragment whichimmunospecifically binds to GITR and/or OX40 (e.g., human GITR and/orOX40) comprising expressing (e.g., recombinantly expressing) theantibody or antigen-binding fragment using a cell or host cell describedherein (e.g., a cell or a host cell comprising polynucleotides encodingan antibody described herein). In a particular embodiment, the cell isan isolated cell. In a particular embodiment, the exogenouspolynucleotides have been introduced into the cell. In a particularembodiment, the method further comprises the step of purifying theantibody or antigen-binding fragment obtained from the cell or hostcell.

Antigen-binding fragments of antibodies can be prepared, e.g., frommonoclonal antibodies, using a wide variety of techniques known in theart including the use of hybridoma, recombinant, and phage displaytechnologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow E & Lane D,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988); Hammerling G J et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. For example, monoclonal antibodies can be producedrecombinantly from host cells exogenously expressing an antibodydescribed herein. Monoclonal antibodies described herein can, forexample, be made by the hybridoma method as described in Kohler G &Milstein C (1975) Nature 256: 495 or can, e.g., be isolated from phagelibraries using the techniques as described herein, for example. Othermethods for the preparation of clonal cell lines and of monoclonalantibodies expressed thereby are well known in the art (see, forexample, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5thEd., Ausubel F M et al., supra).

Further, the antibodies or antigen-binding fragments thereof describedherein can also be generated using various phage display methods knownin the art. In phage display methods, proteins are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In particular, DNA sequences encoding VH and VL domainsare amplified from animal cDNA libraries (e.g., human or murine cDNAlibraries of affected tissues). The DNA encoding the VH and VL domainsare recombined together with a scFv linker by PCR and cloned into aphagemid vector. The vector is electroporated in E. coli and the E. coliis infected with helper phage. Phage used in these methods are typicallyfilamentous phage including fd and M13, and the VH and VL domains areusually recombinantly fused to either the phage gene III or gene VIII.Phage expressing an antibody or fragment that binds to a particularantigen can be selected or identified with antigen, e.g., using labeledantigen or antigen bound or captured to a solid surface or bead.Examples of phage display methods that can be used to make theantibodies described herein include those disclosed in Brinkonan U etal., (1995) J Immunol Methods 182: 41-50; Ames R S et al., (1995) JImmunol Methods 184: 177-186; Kettleborough C A et al., (1994) Eur JImmunol 24: 952-958; Persic L et al., (1997) Gene 187: 9-18; Burton D R& Barbas C F (1994) Advan Immunol 57: 191-280; PCT Application No.PCT/GB91/001134; International Publication Nos. WO 90/02809, WO91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO95/20401, and WO 97/13844; and U.S. Pat. Nos. 5,698,426, 5,223,409,5,403,484, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908,5,516,637, 5,780,225, 5,733,743, and 5,969,108.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate antibodies, including human antibodies, and expressed in anydesired host, including mammalian cells, insect cells, plant cells,yeast, and bacteria, e.g., as described below. Techniques torecombinantly produce antibodies such as Fab, Fab′ and F(ab′)₂ fragmentscan also be employed using methods known in the art such as thosedisclosed in PCT publication No. WO 92/22324; Mullinax R L et al.,(1992) BioTechniques 12(6): 864-9; Sawai H et al., (1995) Am J ReprodImmunol 34: 26-34; and Better M et al., (1988) Science 240: 1041-1043.

In one aspect, to generate antibodies, PCR primers including VH or VLnucleotide sequences, a restriction site, and a flanking sequence toprotect the restriction site can be used to amplify the VH or VLsequences from a template, e.g., scFv clones. Utilizing cloningtechniques known to those of skill in the art, the PCR amplified VHdomains can be cloned into vectors expressing a VH constant region, andthe PCR amplified VL domains can be cloned into vectors expressing a VLconstant region, e.g., human kappa or lambda constant regions. The VHand VL domains can also be cloned into one vector expressing thenecessary constant regions. The heavy chain conversion vectors and lightchain conversion vectors are then co-transfected into cell lines togenerate stable or transient cell lines that express antibodies, e.g.,IgG, using techniques known to those of skill in the art.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules. Forexample, a chimeric antibody can contain a variable region of a mouse orrat monoclonal antibody fused to a constant region of a human antibody.Methods for producing chimeric antibodies are known in the art. See,e.g., Morrison S L (1985) Science 229: 1202-7; Oi V T & Morrison S L(1986) BioTechniques 4: 214-221; Gillies S D et al., (1989) J ImmunolMethods 125: 191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567,4,816,397, and 6,331,415.

A humanized antibody is capable of binding to a predetermined antigenand which comprises a framework region having substantially the aminoacid sequence of a human immunoglobulin and CDRs having substantiallythe amino acid sequence of a non-human immunoglobulin (e.g., a murineimmunoglobulin). In particular embodiments, a humanized antibody alsocomprises at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. The antibody also can includethe CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Ahumanized antibody can be selected from any class of immunoglobulins,including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG₁,IgG₂, IgG₃ and IgG₄. Humanized antibodies can be produced using avariety of techniques known in the art, including but not limited to,CDR-grafting (European Patent No. EP 239400; International PublicationNo. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and5,585,089), veneering or resurfacing (European Patent Nos. EP 592106 andEP 519596; Padlan E A (1991) Mol Immunol 28(4/5): 489-498; Studnicka G Met al., (1994) Prot Engineering 7(6): 805-814; and Roguska M A et al.,(1994) PNAS 91: 969-973), chain shuffling (U.S. Pat. No. 5,565,332), andtechniques disclosed in, e.g., U.S. Pat. Nos. 6,407,213, 5,766,886,International Publication No. WO 93/17105; Tan P et al., (2002) JImmunol 169: 1119-25; Caldas C et al., (2000) Protein Eng. 13(5):353-60; Morea V et al., (2000) Methods 20(3): 267-79; Baca M et al.,(1997) J Biol Chem 272(16): 10678-84; Roguska M A et al., (1996) ProteinEng 9(10): 895 904; Couto J R et al., (1995) Cancer Res. 55 (23 Supp):5973s-5977s; Couto J R et al., (1995) Cancer Res 55(8): 1717-22; SandhuJ S (1994) Gene 150(2): 409-10 and Pedersen J T et al., (1994) J MolBiol 235(3): 959-73. See also U.S. Application Publication No. US2005/0042664 A1 (Feb. 24, 2005), which is incorporated by referenceherein in its entirety.

In particular embodiments, a human antibody comprises an antigen-bindingdomain described herein which binds to the same epitope of GITR or OX40(e.g., human GITR or OX40) as an anti-GITR or OX40 antigen-bindingfragment thereof described herein. In particular embodiments, a humanantibody comprises an antigen-binding fragment which competitivelyblocks (e.g., in a dose-dependent manner) any one of the antigen-bindingfragments described herein, (e.g., pab1949w, pab2049w, pab1876w,pab1967w, pab1975w, or pab1979w) from binding to GITR or OX40 (e.g.,human GITR or OX40). Human antibodies can be produced using any methodknown in the art. For example, transgenic mice which are incapable ofexpressing functional endogenous immunoglobulins, but which can expresshuman immunoglobulin genes, can be used. In particular, the human heavyand light chain immunoglobulin gene complexes can be introduced randomlyor by homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion can be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes can be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of an antigen (e.g., OX40). Monoclonal antibodies directedagainst the antigen can be obtained from the immunized, transgenic miceusing conventional hybridoma technology. The human immunoglobulintransgenes harbored by the transgenic mice rearrange during B celldifferentiation, and subsequently undergo class switching and somaticmutation. Thus, using such a technique, it is possible to producetherapeutically useful IgG, IgA, IgM and IgE antibodies. For an overviewof this technology for producing human antibodies, see Lonberg N &Huszar D (1995) Int Rev Immunol 13:65-93. For a detailed discussion ofthis technology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g.,International Publication Nos. WO 98/24893, WO 96/34096 and WO 96/33735;and U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825,5,661,016, 5,545,806, 5,814,318 and 5,939,598. Examples of mice capableof producing human antibodies include the Xenomouse™ (Abgenix, Inc.;U.S. Pat. Nos. 6,075,181 and 6,150,184), the HuAb-Mouse™ (Mederex,Inc./Gen Pharm; U.S. Pat. Nos. 5,545,806 and 5,569,825), the TransChromo Mouse™ (Kirin) and the KM Mouse™ (Medarex/Kirin).

Human antibodies or antigen-binding fragments which specifically bind toGITR or OX40 (e.g., human GIT or OX40) can be made by a variety ofmethods known in the art including phage display methods described aboveusing antibody libraries derived from human immunoglobulin sequences.See also U.S. Pat. Nos. 4,444,887, 4,716,111, and 5,885,793; andInternational Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO 96/33735, and WO 91/10741.

In some embodiments, human antibodies can be produced using mouse-humanhybridomas. For example, human peripheral blood lymphocytes transformedwith Epstein-Barr virus (EBV) can be fused with mouse myeloma cells toproduce mouse-human hybridomas secreting human monoclonal antibodies,and these mouse-human hybridomas can be screened to determine ones whichsecrete human monoclonal antibodies that immunospecifically bind to atarget antigen (e.g., OX40 (e.g., human OX40)). Such methods are knownand are described in the art, see, e.g., Shinmoto H et al., (2004)Cytotechnology 46: 19-23; Naganawa Y et al., (2005) Human Antibodies 14:27-31.

7.3.1 Polynucleotides

In certain aspects, provided herein are polynucleotides comprising anucleotide sequence encoding an antibody described herein or a fragmentthereof (e.g., a variable light chain region and/or variable heavy chainregion) that immunospecifically binds to a GITR and/or OX40 (e.g., humanGITR and/or OX40) antigen, and vectors, e.g., vectors comprising suchpolynucleotides for recombinant expression in host cells (e.g., E. coliand mammalian cells). Provided herein are polynucleotides comprisingnucleotide sequences encoding any of the antibodies provided herein, aswell as vectors comprising such polynucleotide sequences, e.g.,expression vectors for their efficient expression in host cells, e.g.,mammalian cells.

As used herein, an “isolated” polynucleotide or nucleic acid molecule isone which is separated from other nucleic acid molecules which arepresent in the natural source (e.g., in a mouse or a human) of thenucleic acid molecule. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. For example, the language “substantially free”includes preparations of polynucleotide or nucleic acid molecule havingless than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (in particular lessthan about 10%) of other material, e.g., cellular material, culturemedium, other nucleic acid molecules, chemical precursors and/or otherchemicals. In a specific embodiment, a nucleic acid molecule(s) encodingan antibody described herein is isolated or purified.

In particular aspects, provided herein are polynucleotides comprisingnucleotide sequences encoding antibodies, which immunospecifically bindto a GITR and/or OX40 polypeptide (e.g., human GITR and/or OX40) andcomprises an amino acid sequence as described herein, as well asantibodies that compete with such antibodies for binding to a GITRand/or OX40 polypeptide (e.g., in a dose-dependent manner), or whichbinds to the same epitope as that of such antibodies.

In certain aspects, provided herein are polynucleotides comprising anucleotide sequence encoding the light chain or heavy chain of anantibody described herein. The polynucleotides can comprise nucleotidesequences encoding a light chain or light chain variable domaincomprising the VL CDRs of antibodies described herein (see, e.g., Tables1, 4, and 7). The polynucleotides can comprise nucleotide sequencesencoding a heavy chain or heavy chain variable domain comprising the VHCDRs of antibodies described herein (see, e.g., Tables 1, 5, and 8). Inspecific embodiments, a polynucleotide described herein encodes a VLdomain comprising the amino acid sequence set forth in SEQ ID NO:19, 21,23, 26, 55, or 56. In specific embodiments, a polynucleotide describedherein encodes a VH domain comprising the amino acid sequence set forthin SEQ ID NO:18, 20, 22, 24, 25, and 54.

In particular embodiments, provided herein are polynucleotidescomprising a nucleotide sequence encoding an anti-GITR or OX40antigen-binding domain comprising three VL chain CDRs, e.g., containingVL CDR1, VL CDR2, and VL CDR3 of any one of antibodies described herein(e.g., see Tables 1, 4, and 7). In specific embodiments, provided hereinare polynucleotides comprising three VH chain CDRs, e.g., containing VHCDR1, VH CDR2, and VH CDR3 of any one of antibodies described herein(e.g., see Tables 1, 5, and 8). In specific embodiments, provided hereinare polynucleotides comprising a nucleotide sequence encoding ananti-GITR or OX40 antigen-binding domain comprising three VH chain CDRs,e.g., containing VL CDR1, VL CDR2, and VL CDR3 of any one of antibodiesdescribed herein (e.g., see Tables 1, 4, and 7) and three VH chain CDRs,e.g., containing VH CDR1, VH CDR2, and VH CDR3 of any one of antibodiesdescribed herein (e.g., see Tables 1, 5, and 8).

In certain embodiments, a polynucleotide described herein comprises anucleotide sequence encoding an antibody or antigen-binding domainprovided herein comprising a light chain variable region comprising anamino acid sequence described herein (e.g., SEQ ID NO:19, 21, 23, 26,55, or 56), wherein the antibody or antigen-binding domainimmunospecifically binds to GITR or OX40 (e.g., human GITR or OX40).

In certain embodiments, a polynucleotide described herein comprises anucleotide sequence encoding an antibody or antigen-binding domainprovided herein comprising a heavy chain variable region comprising anamino acid sequence described herein (e.g., SEQ ID NO:18, 20, 22, 24,25, or 54), wherein the antibody or antigen-binding domainimmunospecifically binds to GITR or OX40 (e.g., human GITR or OX40).

In specific aspects, provided herein is a polynucleotide comprising anucleotide sequence encoding an antibody comprising a light chain and aheavy chain, e.g., a separate light chain and heavy chain. With respectto the light chain, in a specific embodiment, a polynucleotide providedherein comprises a nucleotide sequence encoding a kappa light chain. Inanother specific embodiment, a polynucleotide provided herein comprisesa nucleotide sequence encoding a lambda light chain. In yet anotherspecific embodiment, a polynucleotide provided herein comprises anucleotide sequence encoding an antibody described herein comprising ahuman kappa light chain or a human lambda light chain. In a particularembodiment, a polynucleotide provided herein comprises a nucleotidesequence encoding an antibody, which immunospecifically binds to GITRand/or OX40 (e.g., human GITR and/or OX40), wherein the antibodycomprises a light chain, wherein the amino acid sequence of the VLdomain can comprise the amino acid sequence set forth in SEQ ID NO:19,21, 23, 26, 55, or 56 and wherein the constant region of the light chaincomprises the amino acid sequence of a human kappa light chain constantregion. In another particular embodiment, a polynucleotide providedherein comprises a nucleotide sequence encoding an antibody, whichimmunospecifically binds to GITR and/or OX40 (e.g., human GITR and/orOX40), and comprises a light chain, wherein the amino acid sequence ofthe VL domain can comprise the amino acid sequence set forth in SEQ IDNO:19, 21, 23, 26,55, or 56, and wherein the constant region of thelight chain comprises the amino acid sequence of a human lambda lightchain constant region. For example, human constant region sequences canbe those described in U.S. Pat. No. 5,693,780.

In a particular embodiment, a polynucleotide provided herein comprises anucleotide sequence encoding an antibody described herein, whichimmunospecifically binds to GITR and/or OX40 (e.g., human GITR and/orOX40), wherein the antibody comprises a heavy chain, wherein the aminoacid sequence of the VH domain can comprise the amino acid sequence setforth in SEQ ID NO:18, 20, 22, 24, 25, or 54, and wherein the constantregion of the heavy chain comprises the amino acid sequence of a humangamma (γ) heavy chain constant region.

In a certain embodiment, a polynucleotide provided herein comprises anucleotide sequence(s) encoding a VH domain and/or a VL domain of anantibody described herein (such as SEQ ID NO:18, 20, 22, 24, 25, or 54for the VH domain and/or SEQ ID NO:19, 21, 23, 26, 55, or 56 for the VLdomain), which immunospecifically binds to GITR and/or OX40 (e.g., humanOX40).

In yet another specific embodiment, a polynucleotide provided hereincomprises a nucleotide sequence encoding an antibody described herein,which immunospecifically binds GITR and/or OX40 (e.g., human OX40),wherein the antibody comprises a VL domain and a VH domain comprisingany amino acid sequences described herein, wherein the constant regionscomprise the amino acid sequences of the constant regions of a humanIgG₁ (e.g., allotype 1, 17, or 3), human IgG₂, or human IgG₄.

In a specific embodiment, provided herein are polynucleotides comprisinga nucleotide sequence encoding an anti-OX40 antibody or domain thereof,designated herein, see, e.g., Tables 1-9.

Also provided herein are polynucleotides encoding an anti-GITR and/orOX40 antibody or a fragment thereof that are optimized, e.g., bycodon/RNA optimization, replacement with heterologous signal sequences,and elimination of mRNA instability elements. Methods to generateoptimized nucleic acids encoding an anti-GIR and/or OX40 antibody or afragment thereof (e.g., light chain, heavy chain, VH domain, or VLdomain) for recombinant expression by introducing codon changes and/oreliminating inhibitory regions in the mRNA can be carried out byadapting the optimization methods described in, e.g., U.S. Pat. Nos.5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, accordingly.For example, potential splice sites and instability elements (e.g., A/Tor A/U rich elements) within the RNA can be mutated without altering theamino acids encoded by the nucleic acid sequences to increase stabilityof the RNA for recombinant expression. The alterations utilize thedegeneracy of the genetic code, e.g., using an alternative codon for anidentical amino acid. In some embodiments, it can be desirable to alterone or more codons to encode a conservative mutation, e.g., a similaramino acid with similar chemical structure and properties and/orfunction as the original amino acid.

In certain embodiments, an optimized polynucleotide sequence encoding ananti-GITR and/or OX40 antibody described herein or a fragment thereof(e.g., VL domain or VH domain) can hybridize to an antisense (e.g.,complementary) polynucleotide of an unoptimized polynucleotide sequenceencoding an anti-GITR and/or OX40 antibody described herein or afragment thereof (e.g., VL domain or VH domain). In specificembodiments, an optimized nucleotide sequence encoding an anti-GITRand/or OX40 antibody described herein or a fragment hybridizes underhigh stringency conditions to antisense polynucleotide of an unoptimizedpolynucleotide sequence encoding an anti-GITR and/or OX40 antibodydescribed herein or a fragment thereof. In a specific embodiment, anoptimized nucleotide sequence encoding an anti-GITR and/or OX40 antibodydescribed herein or a fragment thereof hybridizes under high stringency,intermediate stringency, or lower stringency hybridization conditions toan antisense polynucleotide of an unoptimized nucleotide sequenceencoding an anti-GITR and/or OX40 antibody described herein or afragment thereof. Information regarding hybridization conditions hasbeen described, see, e.g., U.S. Patent Application Publication No. US2005/0048549 (e.g., paragraphs 72-73), which is incorporated herein byreference.

The DNA also can be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe murine sequences, or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide.

Also provided are polynucleotides that hybridize under high stringency,intermediate or lower stringency hybridization conditions topolynucleotides that encode an antibody described herein. In specificembodiments, polynucleotides described herein hybridize under highstringency, intermediate or lower stringency hybridization conditions topolynucleotides encoding a VH domain (e.g., SEQ ID NO:18, 20, 22, 24,25, 54) and/or VL domain (e.g., SEQ ID NO:19, 21, 23, 25, 55, or 56)provided herein.

Hybridization conditions have been described in the art and are known toone of skill in the art. For example, hybridization under stringentconditions can involve hybridization to filter-bound DNA in 6× sodiumchloride/sodium citrate (SSC) at about 45° C. followed by one or morewashes in 0.2×SSC/0.1% SDS at about 50-65° C.; hybridization underhighly stringent conditions can involve hybridization to filter-boundnucleic acid in 6×SSC at about 45° C. followed by one or more washes in0.1×SSC/0.2% SDS at about 68° C. Hybridization under other stringenthybridization conditions are known to those of skill in the art and havebeen described, see, for example, Ausubel F M et al., eds., (1989)Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York at pages6.3.1-6.3.6 and 2.10.3.

7.3.2 Cells and Vectors

In certain aspects, provided herein are cells (e.g., host cells)expressing (e.g., recombinantly) antibodies described herein whichspecifically bind to GITR and/or OX40 (e.g., human GITR and/or OX40) andrelated polynucleotides and expression vectors. Provided herein arevectors (e.g., expression vectors) comprising polynucleotides comprisingnucleotide sequences encoding anti-GITR and/or OX40 antibodies or afragment for recombinant expression in host cells, preferably inmammalian cells. Also provided herein are host cells comprising suchvectors for recombinantly expressing anti-GITR and/or OX40 antibodiesdescribed herein (e.g., human or humanized antibody). In a particularaspect, provided herein are methods for producing an antibody describedherein, comprising expressing such antibody in a host cell.

Recombinant expression of an antibody or fragment thereof describedherein (e.g., a heavy or light chain of an antibody described herein)that specifically binds to GITR and/or OX40 (e.g., human OX40) involvesconstruction of an expression vector containing a polynucleotide thatencodes the antibody or fragment. Once a polynucleotide encoding anantibody or fragment thereof (e.g., heavy or light chain variabledomains) described herein has been obtained, the vector for theproduction of the antibody molecule can be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody or antibody fragment (e.g., light chain or heavy chain)encoding nucleotide sequence are described herein. Methods which arewell known to those skilled in the art can be used to constructexpression vectors containing antibody or antibody fragment (e.g., lightchain or heavy chain) coding sequences and appropriate transcriptionaland translational control signals. These methods include, for example,in vitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. Also provided are replicable vectors comprising anucleotide sequence encoding an antibody molecule described herein, aheavy or light chain of an antibody, a heavy or light chain variabledomain of an antibody or a fragment thereof, or a heavy or light chainCDR, operably linked to a promoter. Such vectors can, for example,include the nucleotide sequence encoding the constant region of theantibody molecule (see, e.g., International Publication Nos. WO 86/05807and WO 89/01036; and U.S. Pat. No. 5,122,464) and variable domains ofthe antibody can be cloned into such a vector for expression of theentire heavy, the entire light chain, or both the entire heavy and lightchains.

An expression vector can be transferred to a cell (e.g., host cell) byconventional techniques and the resulting cells can then be cultured byconventional techniques to produce an antibody described herein (e.g.,an antibody comprising the CDRs of pab1949w, pab2049w, pab1876w,pab1967w, pab1975w, or pab1979w) or a fragment thereof. Thus, providedherein are host cells containing a polynucleotide encoding an antibodydescribed herein (e.g., an antibody comprising the CDRs of pab1949w,pab2049w, pab1876w, pab1967w, pab1975w, or pab1979w) or fragmentsthereof (e.g., a heavy or light chain thereof, or fragment thereof),operably linked to a promoter for expression of such sequences in thehost cell. In certain embodiments, for the expression of double-chainedantibodies, vectors encoding both the heavy and light chains,individually, can be co-expressed in the host cell for expression of theentire immunoglobulin molecule, as detailed below. In certainembodiments, a host cell contains a vector comprising a polynucleotideencoding both the heavy chain and light chain of an antibody describedherein (e.g., an antibody comprising the CDRs of pab1949w, pab2049w,pab1876w, pab1967w, pab1975w, or pab1979w), or a fragment thereof. Inspecific embodiments, a host cell contains two different vectors, afirst vector comprising a polynucleotide encoding a heavy chain or aheavy chain variable region of an antibody described herein (e.g., anantibody comprising the CDRs of pab1949w, pab2049w, pab1876w, pab1967w,pab1975w, or pab1979w), or a fragment thereof, and a second vectorcomprising a polynucleotide encoding a light chain or a light chainvariable region of an antibody described herein (e.g., an antibodycomprising the CDRs of pab1949w, pab2049w, pab1876w, pab1967w, pab1975w,or pab1979w), or a fragment thereof. In other embodiments, a first hostcell comprises a first vector comprising a polynucleotide encoding aheavy chain or a heavy chain variable region of an antibody describedherein (e.g., an antibody comprising the CDRs of pab1949w, pab2049w,pab1876w, pab1967w, pab1975w, or pab1979w), or a fragment thereof, and asecond host cell comprises a second vector comprising a polynucleotideencoding a light chain or a light chain variable region of an antibodydescribed herein (e.g., an antibody comprising the CDRs of pab1949w,pab2049w, pab1876w, pab1967w, pab1975w, or pab1979w). In specificembodiments, a heavy chain/heavy chain variable region expressed by afirst cell associated with a light chain/light chain variable region ofa second cell to form an anti-GITR and/or OX40 antibody described herein(e.g., antibody comprising the CDRs pab1949w, pab2049w, pab1876w,pab1967w, pab1975w, or pab1979w). In certain embodiments, providedherein is a population of host cells comprising such first host cell andsuch second host cell.

In a particular embodiment, provided herein is a population of vectorscomprising a first vector comprising a polynucleotide encoding a lightchain/light chain variable region of an anti-GITR and/or OX40 antibodydescribed herein (e.g., antibody comprising the CDRs of pab1949w,pab2049w, pab1876w, pab1967w, pab1975w, or pab1979w), and a secondvector comprising a polynucleotide encoding a heavy chain/heavy chainvariable region of an anti-OX40 antibody described herein (e.g.,antibody comprising the CDRs of pab1949w, pab2049w, pab1876w, pab1967w,pab1975w, or pab1979w).

A variety of host-expression vector systems can be utilized to expressantibody molecules described herein (see, e.g., U.S. Pat. No.5,807,715). Such host-expression systems represent vehicles by which thecoding sequences of interest can be produced and subsequently purified,but also represent cells which can, when transformed or transfected withthe appropriate nucleotide coding sequences, express an antibodymolecule described herein in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli and B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing antibody coding sequences; yeast(e.g., Saccharomyces Pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing antibody coding sequences; plant cell systems(e.g., green algae such as Chlamydomonas reinhardtii) infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS),CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, andNIH 3T3, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20 andBMT10 cells) harboring recombinant expression constructs containingpromoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). In aspecific embodiment, cells for expressing antibodies described herein(e.g., an antibody comprising the CDRs of any one of antibodiespab1949w, pab2049w, pab1876w, pab1967w, pab1975w, or pab1979w) are CHOcells, for example CHO cells from the CHO GS System™ (Lonza). In aparticular embodiment, cells for expressing antibodies described hereinare human cells, e.g., human cell lines. In a specific embodiment, amammalian expression vector is pOptiVEC™ or pcDNA3.3. In a particularembodiment, bacterial cells such as Escherichia coli, or eukaryoticcells (e.g., mammalian cells), especially for the expression of wholerecombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary (CHO) cells in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking M K & Hofstetter H (1986) Gene 45: 101-105; and Cockett M I etal., (1990) Biotechnology 8: 662-667). In certain embodiments,antibodies described herein are produced by CHO cells or NS0 cells. In aspecific embodiment, the expression of nucleotide sequences encodingantibodies described herein which immunospecifically bind GITR and/orOX40 (e.g., human GITR and/or OX40) is regulated by a constitutivepromoter, inducible promoter or tissue specific promoter.

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, Hela, MDCK, HEK293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murinemyeloma cell line that does not endogenously produce any immunoglobulinchains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst,HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20, BMT10 andHsS78Bst cells. In certain embodiments, anti-GITR and/or OX40 antibodiesdescribed herein are produced in mammalian cells, such as CHO cells.

In a specific embodiment, the antibodies described herein have reducedfucose content or no fucose content. Such antibodies can be producedusing techniques known one skilled in the art. For example, theantibodies can be expressed in cells deficient or lacking the ability ofto fucosylate. In a specific example, cell lines with a knockout of bothalleles of α1,6-fucosyltransferase can be used to produce antibodieswith reduced fucose content. The Potelligent® system (Lonza) is anexample of such a system that can be used to produce antibodies withreduced fucose content.

For long-term, high-yield production of recombinant proteins, stableexpression cells can be generated. For example, cell lines which stablyexpress an anti-GITR and/or OX40 antibody described herein can beengineered.

Once an antibody molecule described herein has been produced byrecombinant expression, it can be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theantibodies described herein can be fused to heterologous polypeptidesequences described herein or otherwise known in the art to facilitatepurification.

In specific embodiments, an antibody described herein is isolated orpurified. Generally, an isolated antibody is one that is substantiallyfree of other antibodies with different antigenic specificities than theisolated antibody. For example, in a particular embodiment, apreparation of an antibody described herein is substantially free ofcellular material and/or chemical precursors. The language“substantially free of cellular material” includes preparations of anantibody in which the antibody is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. Thus, anantibody that is substantially free of cellular material includespreparations of antibody having less than about 30%, 20%, 10%, 5%, 2%,1%, 0.5%, or 0.1% (by dry weight) of heterologous protein (also referredto herein as a “contaminating protein”) and/or variants of an antibody,for example, different post-translational modified forms of an antibody.When the antibody or fragment is recombinantly produced, it is alsogenerally substantially free of culture medium, i.e., culture mediumrepresents less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volumeof the protein preparation. When the antibody or fragment is produced bychemical synthesis, it is generally substantially free of chemicalprecursors or other chemicals, i.e., it is separated from chemicalprecursors or other chemicals which are involved in the synthesis of theprotein. Accordingly, such preparations of the antibody or fragment haveless than about 30%, 20%, 10%, or 5% (by dry weight) of chemicalprecursors or compounds other than the antibody or fragment of interest.In a specific embodiment, antibodies described herein are isolated orpurified.

7.4 Pharmaceutical Compositions

Provided herein are compositions comprising an antibody described hereinhaving the desired degree of purity in a physiologically acceptablecarrier, excipient or stabilizer (Remington's Pharmaceutical Sciences(1990) Mack Publishing Co., Easton, PA). Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed.

Pharmaceutical compositions described herein that comprise an agonisticantibody described herein can be useful in enhancing, inducing, oractivating a GITR and/or OX40 activity and treating a condition, such ascancer or an infectious disease. Examples of cancer that can be treatedin accordance with the methods described herein include, but are notlimited to, B cell lymphomas (e.g., B cell chronic lymphocytic leukemia,B cell non-Hodgkin's lymphoma, cutaneous B cell lymphoma, diffuse largeB cell lymphoma), basal cell carcinoma, bladder cancer, blastoma, brainmetastasis, breast cancer, Burkitt lymphoma, carcinoma (e.g.,adenocarcinoma (e.g., of the gastroesophageal junction)), cervicalcancer, colon cancer, colorectal cancer (colon cancer and rectalcancer), endometrial carcinoma, esophageal cancer, Ewing sarcoma,follicular lymphoma, gastric cancer, gastroesophageal junctioncarcinoma, gastrointestinal cancer, glioblastoma (e.g., glioblastomamultiforme, e.g., newly diagnosed or recurrent), glioma, head and neckcancer (e.g., head and neck squamous cell carcinoma), hepaticmetastasis, Hodgkin's and non-Hodgkin's lymphoma, kidney cancer (e.g.,renal cell carcinoma and Wilms' tumors), laryngeal cancer, leukemia(e.g., chronic myelocytic leukemia, hairy cell leukemia), liver cancer(e.g., hepatic carcinoma and hepatoma), lung cancer (e.g., non-smallcell lung cancer and small-cell lung cancer), lymphblastic lymphoma,lymphoma, mantle cell lymphoma, metastatic brain tumor, metastaticcancer, myeloma (e.g., multiple myeloma), neuroblastoma, ocularmelanoma, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreaticcancer (e.g., pancreatis ductal adenocarcinoma), prostate cancer (e.g.,hormone refractory (e.g., castration resistant), metastatic, metastatichormone refractory (e.g., castration resistant, androgen independent)),renal cell carcinoma (e.g., metastatic), salivary gland carcinoma,sarcoma (e.g., rhabdomyosarcoma), skin cancer (e.g., melanoma (e.g.,metastatic melanoma)), soft tissue sarcoma, solid tumor, squamous cellcarcinoma, synovia sarcoma, testicular cancer, thyroid cancer,transitional cell cancer (urothelial cell cancer), uveal melanoma (e.g.,metastatic), verrucous carcinoma, vulval cancer, and Waldenstrommacroglobulinemia.

Pharmaceutical compositions described herein that comprise anantagonistic antibody described herein can be useful in reducing,inhibiting, or deactivating a GITR and/or OX40 activity and treating acondition, such as an inflammatory or autoimmune disease or disorder oran infectious disease.

Pharmaceutical compositions described herein that comprise anantagonistic antibody described herein can be useful in reducing,deactivating, or inhibiting GITR and/or OX40 activity and treating acondition selected from the group consisting of infections (viral,bacterial, fungal and parasitic), endotoxic shock associated withinfection, arthritis, rheumatoid arthritis, asthma, chronic obstructivepulmonary disease (COPD), pelvic inflammatory disease, Alzheimer'sDisease, inflammatory bowel disease, Crohn's disease, ulcerativecolitis, Peyronie's Disease, coeliac disease, gallbladder disease,Pilonidal disease, peritonitis, psoriasis, vasculitis, surgicaladhesions, stroke, Type I Diabetes, lyme disease, arthritis,meningoencephalitis, uveitis, autoimmune uveitis, immune mediatedinflammatory disorders of the central and peripheral nervous system suchas multiple sclerosis, lupus (such as systemic lupus erythematosus) andGuillain-Barr syndrome, dermatitis, Atopic dermatitis, autoimmunehepatitis, fibrosing alveolitis, Grave's disease, IgA nephropathy,idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus,primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener'sgranulomatosis, pancreatitis, trauma (surgery), graft-versus-hostdisease, transplant rejection, heart disease (i.e., cardiovasculardisease) including ischaemic diseases such as myocardial infarction aswell as atherosclerosis, intravascular coagulation, bone resorption,osteoporosis, osteoarthritis, periodontitis, hypochlorhydia, andneuromyelitis optica.

The compositions to be used for in vivo administration can be sterile.This is readily accomplished by filtration through, e.g., sterilefiltration membranes.

7.5 Uses and Methods 7.5.1 Therapeutic Uses and Methods

In one aspect, presented herein are methods for modulating one or moreimmune functions or responses in a subject, comprising to a subject inneed thereof administering a multispecific (e.g., bispecific) antibodythat binds to GITR and/or OX40 described herein, or a compositioncomprising such an antibody. In a specific aspect, presented herein aremethods for activating, enhancing or inducing one or more immunefunctions or responses in a subject, comprising administering to asubject in need thereof a multispecific (e.g., bispecific) antibody thatbinds to GITR and/or OX40 or a composition comprising such an antibody.In a specific embodiment, presented herein are methods for preventingand/or treating diseases in which it is desirable to activate or enhanceone or more immune functions or responses, comprising administering to asubject in need thereof a multispecific (e.g., bispecific) antibody thatbinds to GITR and/or OX40 described herein or a composition thereof. Ina certain embodiment, presented herein are methods of treating aninfectious disease comprising administering to a subject in need thereofa multispecific (e.g., bispecific) antibody that binds to GITR and/orOX40 or a composition thereof. In a certain embodiment, presented hereinare methods of treating cancer comprising administering to a subject inneed thereof a multispecific (e.g., bispecific) antibody that binds toGITR and/or OX40 or a composition thereof. The cancer can be selectedfrom a group consisting of melanoma, renal cancer, and prostate cancer.The cancer can be selected from a group consisting of melanoma, renalcancer, prostate cancer, colon cancer, and lung cancer. In a certainembodiment, presented herein are methods of treating melanoma comprisingadministering to a subject in need thereof a multispecific (e.g.,bispecific) antibody that binds to GITR and/or OX40 or a compositionthereof. In a certain embodiment, presented herein are methods oftreating renal cancer comprising administering to a subject in needthereof a multispecific (e.g., bispecific) antibody that binds to GITRand/or OX40 or a composition thereof. In a certain embodiment, presentedherein are methods of treating prostate cancer comprising administeringto a subject in need thereof a multispecific (e.g., bispecific) antibodythat binds to GITR and/or OX40 or a composition thereof. In certainembodiments, presented herein are methods of treating colon cancercomprising administering to a subject in need thereof a multispecific(e.g., bispecific) antibody that binds to GITR and/or OX40 or acomposition thereof. In certain embodiments, presented herein aremethods of treating lung cancer comprising administering to a subject inneed thereof a multispecific (e.g., bispecific) antibody that binds toGITR and/or OX40 or a composition thereof. In certain embodiments,presented herein are methods of treating non-small cell lung cancer(NSCLC) comprising administering to a subject in need thereof amultispecific (e.g., bispecific) antibody that binds to GITR and/or OX40or a composition thereof.

In a certain embodiment, presented herein are methods of treating acancer selected from the group consisting of: B cell lymphomas (e.g., Bcell chronic lymphocytic leukemia, B cell non-Hodgkin's lymphoma,cutaneous B cell lymphoma, diffuse large B cell lymphoma), basal cellcarcinoma, bladder cancer, blastoma, brain metastasis, breast cancer,Burkitt lymphoma, carcinoma (e.g., adenocarcinoma (e.g., of thegastroesophageal junction)), cervical cancer, colon cancer, colorectalcancer (colon cancer and rectal cancer), endometrial carcinoma,esophageal cancer, Ewing sarcoma, follicular lymphoma, gastric cancer,gastroesophageal junction carcinoma, gastrointestinal cancer,glioblastoma (e.g., glioblastoma multiforme, e.g., newly diagnosed orrecurrent), glioma, head and neck cancer (e.g., head and neck squamouscell carcinoma), hepatic metastasis, Hodgkin's and non-Hodgkin'slymphoma, kidney cancer (e.g., renal cell carcinoma and Wilms' tumors),laryngeal cancer, leukemia (e.g., chronic myelocytic leukemia, hairycell leukemia), liver cancer (e.g., hepatic carcinoma and hepatoma),lung cancer (e.g., non-small cell lung cancer and small-cell lungcancer), lymphblastic lymphoma, lymphoma, mantle cell lymphoma,metastatic brain tumor, metastatic cancer, myeloma (e.g., multiplemyeloma), neuroblastoma, ocular melanoma, oropharyngeal cancer,osteosarcoma, ovarian cancer, pancreatic cancer (e.g., pancreatis ductaladenocarcinoma), prostate cancer (e.g., hormone refractory (e.g.,castration resistant), metastatic, metastatic hormone refractory (e.g.,castration resistant, androgen independent)), renal cell carcinoma(e.g., metastatic), salivary gland carcinoma, sarcoma (e.g.,rhabdomyosarcoma), skin cancer (e.g., melanoma (e.g., metastaticmelanoma)), soft tissue sarcoma, solid tumor, squamous cell carcinoma,synovia sarcoma, testicular cancer, thyroid cancer, transitional cellcancer (urothelial cell cancer), uveal melanoma (e.g., metastatic),verrucous carcinoma, vulval cancer, and Waldenstrom macroglobulinemia.

In another embodiment, a multispecific (e.g., bispecific) antibody thatbinds to GITR and/or OX40 is administered to a patient diagnosed withcancer to increase the proliferation and/or effector function of one ormore immune cell populations (e.g., T cell effector cells, such as CD4⁺and CD8⁺ T cells) in the patient.

In a specific embodiment, a multispecific (e.g., bispecific) antibodythat binds to GITR and/or OX40 described herein activates or enhances orinduces one or more immune functions or responses in a subject by atleast 99%, at least 98%, at least 95%, at least 90%, at least 85%, atleast 80%, at least 75%, at least 70%, at least 60%, at least 50%, atleast 45%, at least 40%, at least 45%, at least 35%, at least 30%, atleast 25%, at least 20%, or at least 10%, or in the range of between 10%to 25%, 25% to 50%, 50% to 75%, or 75% to 95% relative to the immunefunction in a subject not administered the multispecific (e.g.,bispecific) antibody that binds to GITR and/or OX40 described hereinusing assays well known in the art, e.g., ELISPOT, ELISA, and cellproliferation assays. In a specific embodiment, the immune function iscytokine production (e.g., IL-2, TNF-α, IFN-γ, IL-4, IL-10, and/or IL-13production). In another embodiment, the immune function is T cellproliferation/expansion, which can be assayed, e.g., by flow cytometryto detect the number of cells expressing markers of T cells (e.g., CD3,CD4, or CD8). In another embodiment, the immune function is antibodyproduction, which can be assayed, e.g., by ELISA. In some embodiments,the immune function is effector function, which can be assayed, e.g., bya cytotoxicity assay or other assays well known in the art. In anotherembodiment, the immune function is a Th1 response. In anotherembodiment, the immune function is a Th2 response. In anotherembodiment, the immune function is a memory response.

In specific embodiments, non-limiting examples of immune functions thatcan be enhanced or induced by a multispecific (e.g., bispecific)antibody that binds to GITR and/or OX40 are proliferation/expansion ofeffector lymphocytes (e.g., increase in the number of effector Tlymphocytes), and inhibition of apoptosis of effector lymphocytes (e.g.,effector T lymphocytes). In particular embodiments, an immune functionenhanced or induced by a multispecific (e.g., bispecific) antibody thatbinds to GITR and/or OX40 described herein is proliferation/expansion inthe number of or activation of CD4⁺ T cells (e.g., Th1 and Th2 helper Tcells), CD8⁺ T cells (e.g., cytotoxic T lymphocytes, alpha/beta T cells,and gamma/delta T cells), B cells (e.g., plasma cells), memory T cells,memory B cells, tumor-resident T cells, CD122⁺ T cells, natural killer(NK) cells), macrophages, monocytes, dendritic cells, mast cells,eosinophils, basophils or polymorphonucleated leukocytes. In oneembodiment, a multispecific (e.g., bispecific) antibody that binds toGITR and/or OX40 described herein activates or enhances theproliferation/expansion or number of lymphocyte progenitors. In someembodiments, a multispecific (e.g., bispecific) antibody that binds toGITR and/or OX40 described herein increases the number of CD4⁺ T cells(e.g., Th1 and Th2 helper T cells), CD8⁺ T cells (e.g., cytotoxic Tlymphocytes, alpha/beta T cells, and gamma/delta T cells), B cells(e.g., plasma cells), memory T cells, memory B cells, tumor-resident Tcells, CD122⁺ T cells, natural killer cells (NK cells), macrophages,monocytes, dendritic cells, mast cells, eosinophils, basophils orpolymorphonucleated leukocytes by approximately at least 99%, at least98%, at least 95%, at least 90%, at least 85%, at least 80%, at least75%, at least 70%, at least 60%, at least 50%, at least 45%, at least40%, at least 45%, at least 35%, at least 30%, at least 25%, at least20%, or at least 10%, or in the range of between 10% to 25%, 25% to 50%,50% to 75%, or 75% to 95% relative a negative control (e.g., number ofthe respective cells not treated, cultured, or contacted with amultispecific (e.g., bispecific) antibody that binds to GITR and/or OX40described herein).

In some embodiments, a multispecific (e.g., bispecific) antibody thatbinds to GITR and/or OX40 described herein is administered to a subjectin combination with a compound that targets an immunomodulatoryenzyme(s) such as IDO (indoleamine-(2,3)-dioxygenase) and TDO(tryptophan 2,3-dioxygenase). In particular embodiments, such compoundis selected from the group consisting of epacadostat (Incyte Corp),F001287 (Flexus Biosciences), indoximod (NewLink Genetics), and NLG919(NewLink Genetics). In one embodiment, the compound is epacadostat. Inanother embodiment, the compound is F001287. In another embodiment, thecompound is indoximod. In another embodiment, the compound is NLG919.

In some embodiments, an a multispecific (e.g., bispecific) antibody thatbinds to GITR and/or OX40 described herein is administered to a subjectin combination with a vaccine.

In some embodiments, a multispecific (e.g., bispecific) antibody thatbinds to GITR and/or OX40 described herein is administered to a subjectin combination with a heat shock protein based tumor vaccine or a heatshock protein based pathogen vaccine. In a specific embodiment, amultispecific (e.g., bispecific) antibody that binds to GITR and/or OX40is administered to a subject in combination with a heat shock proteinbased tumor-vaccine. Heat shock proteins (HSPs) are a family of highlyconserved proteins found ubiquitously across all species. Theirexpression can be powerfully induced to much higher levels as a resultof heat shock or other forms of stress, including exposure to toxins,oxidative stress or glucose deprivation. Five families have beenclassified according to molecular weight: HSP-110, -90, -70, -60 and-28. HSPs deliver immunogenic peptides through the cross-presentationpathway in antigen presenting cells (APCs) such as macrophages anddendritic cells (DCs), leading to T cell activation. HSPs function aschaperone carriers of tumor-associated antigenic peptides formingcomplexes able to induce tumor-specific immunity. Upon release fromdying tumor cells, the HSP-antigen complexes are taken up byantigen-presenting cells (APCs) wherein the antigens are processed intopeptides that bind MHC class I and class II molecules leading to theactivation of anti-tumor CD8+ and CD4+ T cells. The immunity elicited byHSP complexes derived from tumor preparations is specifically directedagainst the unique antigenic peptide repertoire expressed by the cancerof each subject.

A heat shock protein peptide complex (HSPPC) is a protein peptidecomplex consisting of a heat shock protein non-covalently complexed withantigenic peptides. HSPPCs elicit both innate and adaptive immuneresponses. In a specific embodiment, the antigenic peptide(s) displaysantigenicity for the cancer being treated. HSPPCs are efficiently seizedby APCs via membrane receptors (mainly CD91) or by binding to Toll-likereceptors. HSPPC internalization results in functional maturation of theAPCs with chemokine and cytokine production leading to activation ofnatural killer cells (NK), monocytes and Th1 and Th-2-mediated immuneresponses. In some embodiments, HSPPCs used in methods disclosed hereincomprise one or more heat shock proteins from the hsp60, hsp70, or hsp90family of stress proteins complexed with antigenic peptides. In someembodiments, HSPPCs comprise hsc70, hsp70, hsp90, hsp110, grp170, gp96,calreticulin, or combinations of two or more thereof.

In a specific embodiment, a multispecific (e.g., bispecific) antibodythat binds to GITR and/or OX40 is administered to a subject incombination with a heat shock protein peptide complex (HSPPC), e.g.,heat shock protein peptide complex-96 (HSPPC-96), to treat cancer.HSPPC-96 comprises a 96 kDa heat shock protein (Hsp), gp96, complexed toantigenic peptides. HSPPC-96 is a cancer immunotherapy manufactured froma subject's tumor and contains the cancer's antigenic “fingerprint.” Insome embodiments, this fingerprint contains unique antigens that arepresent only in that particular subject's specific cancer cells andinjection of the vaccine is intended to stimulate the subject's immunesystem to recognize and attack any cells with the specific cancerfingerprint.

In some embodiments, the HSPPC, e.g., HSPPC-96, is produced from thetumor tissue of a subject. In a specific embodiment, the HSPPC (e.g.,HSPPC-96) is produced from tumor of the type of cancer or metastasisthereof being treated. In another specific embodiment, the HSPPC (e.g.,HSPPC-96) is autologous to the subject being treated. In someembodiments, the tumor tissue is non-necrotic tumor tissue. In someembodiments, at least 1 gram (e.g., at least 1, at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,or at least 10 grams) of non-necrotic tumor tissue is used to produce avaccine regimen. In some embodiments, after surgical resection,non-necrotic tumor tissue is frozen prior to use in vaccine preparation.In some embodiments, the HSPPC, e.g., HSPPC-96, is isolated from thetumor tissue by purification techniques, filtered and prepared for aninjectable vaccine. In some embodiments, a subject is administered 6-12doses of the HSPPC, e.g., HSPCC-96. In such embodiments, the HSPPC,e.g., HSPPC-96, doses may be administered weekly for the first 4 dosesand then biweekly for the 2-8 additional doses.

Further examples of HSPPCs that may be used in accordance with themethods described herein are disclosed in the following patents andpatent applications, which are incorporated herein by reference in theirentireties for all purposes, U.S. Pat. Nos. 6,391,306, 6,383,492,6,403,095, 6,410,026, 6,436,404, 6,447,780, 6,447,781 and 6,610,659.

In one aspect, the methods for modulating one or more immune functionsor responses in a subject as presented herein are methods fordeactivating, reducing or inhibiting one or more immune functions orresponses in a subject, comprising to a subject in need thereofadministering an antagonistic multispecific (e.g., bispecific) antibodythat binds to GITR and/or OX40 or a composition thereof. In a specificembodiment, presented herein are methods for preventing and/or treatingdiseases in which it is desirable to deactivate, reduce, or inhibit oneor more immune functions or responses, comprising administering to asubject in need thereof an antagonistic multispecific (e.g., bispecific)antibody that binds to GITR and/or OX40 or a composition thereof.

In a certain embodiment, presented herein are methods of treating anautoimmune or inflammatory disease or disorder comprising administeringto a subject in need thereof an effective amount of an antagonisticmultispecific (e.g., bispecific) antibody that binds to GITR and/or OX40or a composition thereof. In certain embodiments, the disease ordisorder is selected from the group consisting of: infections (viral,bacterial, fungal and parasitic), endotoxic shock associated withinfection, arthritis, rheumatoid arthritis, asthma, chronic obstructivepulmonary disease (COPD), pelvic inflammatory disease, Alzheimer'sDisease, inflammatory bowel disease, Crohn's disease, ulcerativecolitis, Peyronie's Disease, coeliac disease, gallbladder disease,Pilonidal disease, peritonitis, psoriasis, vasculitis, surgicaladhesions, stroke, Type I Diabetes, lyme disease, arthritis,meningoencephalitis, uveitis, autoimmune uveitis, immune mediatedinflammatory disorders of the central and peripheral nervous system suchas multiple sclerosis, lupus (such as systemic lupus erythematosus) andGuillain-Barr syndrome, dermatitis, Atopic dermatitis, autoimmunehepatitis, fibrosing alveolitis, Grave's disease, IgA nephropathy,idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus,primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener'sgranulomatosis, pancreatitis, trauma (surgery), graft-versus-hostdisease, transplant rejection, heart disease (i.e., cardiovasculardisease) including ischaemic diseases such as myocardial infarction aswell as atherosclerosis, intravascular coagulation, bone resorption,osteoporosis, osteoarthritis, periodontitis, hypochlorhydia, andneuromyelitis optica. In certain embodiments, the autoimmune orinflammatory disease or disorder is transplant rejection,graft-versus-host disease, vasculitis, asthma, rheumatoid arthritis,dermatitis, inflammatory bowel disease, uveitis, lupus, colitis,diabetes, multiple sclerosis, or airway inflammation. In certainembodiments, the subject is a human.

In another embodiment, an antagonistic multispecific (e.g., bispecific)antibody that binds to GITR and/or OX40 is administered to a patientdiagnosed with an autoimmune or inflammatory disease or disorder todecrease the proliferation and/or effector function of one or moreimmune cell populations (e.g., T cell effector cells, such as CD4⁺ andCD8⁺ T cells) in the patient. In certain embodiments, the autoimmune orinflammatory disease or disorder is transplant rejection,graft-versus-host disease, vasculitis, asthma, rheumatoid arthritis,dermatitis, inflammatory bowel disease, uveitis, lupus, colitis,diabetes, multiple sclerosis, or airway inflammation.

In a specific embodiment, an antagonistic multispecific (e.g.,bispecific) antibody that binds to GITR and/or OX40 described hereindeactivates or reduces or inhibits one or more immune functions orresponses in a subject by at least 99%, at least 98%, at least 95%, atleast 90%, at least 85%, at least 80%, at least 75%, at least 70%, atleast 60%, at least 50%, at least 45%, at least 40%, at least 45%, atleast 35%, at least 30%, at least 25%, at least 20%, or at least 10%, orin the range of between 10% to 25%, 25% to 50%, 50% to 75%, or 75% to95% relative to the immune function in a subject not administered theantagonistic multispecific (e.g., bispecific) antibody that binds toGITR and/or OX40 described herein using assays well known in the art,e.g., ELISPOT, ELISA, and cell proliferation assays. In a specificembodiment, the immune function is cytokine production (e.g., IL-2,TNF-α, IFN-γ, IL-4, IL-10, and/or IL-13 production). In anotherembodiment, the immune function is T cell proliferation/expansion, whichcan be assayed, e.g., by flow cytometry to detect the number of cellsexpressing markers of T cells (e.g., CD3, CD4, or CD8). In anotherembodiment, the immune function is antibody production, which can beassayed, e.g., by ELISA. In some embodiments, the immune function iseffector function, which can be assayed, e.g., by a cytotoxicity assayor other assays well known in the art. In another embodiment, the immunefunction is a Th1 response. In another embodiment, the immune functionis a Th2 response. In another embodiment, the immune function is amemory response.

In specific embodiments, non-limiting examples of immune functions thatcan be reduced or inhibited by a an antagonistic multispecific (e.g.,bispecific) antibody that binds to GITR and/or OX40 areproliferation/expansion of effector lymphocytes (e.g., decrease in thenumber of effector T lymphocytes), and stimulation of apoptosis ofeffector lymphocytes (e.g., effector T lymphocytes). In particularembodiments, an immune function reduced or inhibited by an antagonisticmultispecific (e.g., bispecific) antibody that binds to GITR and/or OX40described herein is proliferation/expansion in the number of oractivation of CD4⁺ T cells (e.g., Th1 and Th2 helper T cells), CD8⁺ Tcells (e.g., cytotoxic T lymphocytes, alpha/beta T cells, andgamma/delta T cells), B cells (e.g., plasma cells), memory T cells,memory B cells, tumor-resident T cells, CD122⁺ T cells, natural killer(NK) cells), macrophages, monocytes, dendritic cells, mast cells,eosinophils, basophils or polymorphonucleated leukocytes. In oneembodiment, an antagonistic multispecific (e.g., bispecific) antibodythat binds to GITR and/or OX40 described herein deactivates or reducesthe proliferation/expansion or number of lymphocyte progenitors. In someembodiments, an antagonistic multispecific (e.g., bispecific) antibodythat binds to GITR and/or OX40 described herein decreases the number ofCD4⁺ T cells (e.g., Th1 and Th2 helper T cells), CD8⁺ T cells (e.g.,cytotoxic T lymphocytes, alpha/beta T cells, and gamma/delta T cells), Bcells (e.g., plasma cells), memory T cells, memory B cells,tumor-resident T cells, CD122⁺ T cells, natural killer cells (NK cells),macrophages, monocytes, dendritic cells, mast cells, eosinophils,basophils or polymorphonucleated leukocytes by approximately at least99%, at least 98%, at least 95%, at least 90%, at least 85%, at least80%, at least 75%, at least 70%, at least 60%, at least 50%, at least45%, at least 40%, at least 45%, at least 35%, at least 30%, at least25%, at least 20%, or at least 10%, or in the range of between 10% to25%, 25% to 50%, 50% to 75%, or 75% to 95% relative a negative control(e.g., number of the respective cells not treated, cultured, orcontacted with an antagonistic multispecific (e.g., bispecific) antibodythat binds to GITR and/or OX40 antibody described herein).

7.5.1.1 Routes of Administration & Dosage

An antibody or composition described herein can be delivered to asubject by a variety of routes.

The amount of an antibody or composition which will be effective in thetreatment and/or prevention of a condition will depend on the nature ofthe disease, and can be determined by standard clinical techniques.

The precise dose to be employed in a composition will also depend on theroute of administration, and the seriousness of the disease, and shouldbe decided according to the judgment of the practitioner and eachsubject's circumstances. For example, effective doses may also varydepending upon means of administration, target site, physiological stateof the patient (including age, body weight and health), whether thepatient is human or an animal, other medications administered, orwhether treatment is prophylactic or therapeutic. Usually, the patientis a human but non-human mammals including transgenic mammals can alsobe treated. Treatment dosages are optimally titrated to optimize safetyand efficacy.

In certain embodiments, an in vitro assay is employed to help identifyoptimal dosage ranges. Effective doses may be extrapolated from doseresponse curves derived from in vitro or animal model test systems.

Generally, human antibodies have a longer half-life within the humanbody than antibodies from other species due to the immune response tothe foreign polypeptides. Thus, lower dosages of human antibodies andless frequent administration is often possible.

7.5.2 Detection & Diagnostic Uses

An anti-OX40 antibody described herein (see, e.g., Section 7.2) can beused to assay OX40 protein levels in a biological sample using classicalimmunohistological methods known to those of skill in the art, includingimmunoassays, such as the enzyme linked immunosorbent assay (ELISA),immunoprecipitation, or Western blotting. Suitable antibody assay labelsare known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (¹²⁵I, ¹²¹I) carbon (¹⁴C), sulfur(³⁵S), tritium (³H), indium (¹²¹In), and technetium (⁹⁹Tc); luminescentlabels, such as luminol; and fluorescent labels, such as fluorescein andrhodamine, and biotin. Such labels can be used to label an antibodydescribed herein. Alternatively, a second antibody that recognizes ananti-OX40 antibody described herein can be labeled and used incombination with an anti-OX40 antibody to detect OX40 protein levels.

Assaying for the expression level of OX40 protein is intended to includequalitatively or quantitatively measuring or estimating the level of aOX40 protein in a first biological sample either directly (e.g., bydetermining or estimating absolute protein level) or relatively (e.g.,by comparing to the disease associated protein level in a secondbiological sample). OX40 polypeptide expression level in the firstbiological sample can be measured or estimated and compared to astandard OX40 protein level, the standard being taken from a secondbiological sample obtained from an individual not having the disorder orbeing determined by averaging levels from a population of individualsnot having the disorder. As will be appreciated in the art, once the“standard” OX40 polypeptide level is known, it can be used repeatedly asa standard for comparison.

As used herein, the term “biological sample” refers to any biologicalsample obtained from a subject, cell line, tissue, or other source ofcells potentially expressing OX40. Methods for obtaining tissue biopsiesand body fluids from animals (e.g., humans) are well known in the art.Biological samples include peripheral mononuclear blood cells.

An anti-OX40 antibody described herein can be used for prognostic,diagnostic, monitoring and screening applications, including in vitroand in vivo applications well known and standard to the skilled artisanand based on the present description. Prognostic, diagnostic, monitoringand screening assays and kits for in vitro assessment and evaluation ofimmune system status and/or immune response may be utilized to predict,diagnose and monitor to evaluate patient samples including those knownto have or suspected of having an immune system-dysfunction or withregard to an anticipated or desired immune system response, antigenresponse or vaccine response. The assessment and evaluation of immunesystem status and/or immune response is also useful in determining thesuitability of a patient for a clinical trial of a drug or for theadministration of a particular chemotherapeutic agent or an antibody,including combinations thereof, versus a different agent or antibody.This type of prognostic and diagnostic monitoring and assessment isalready in practice utilizing antibodies against the HER2 protein inbreast cancer (HercepTest™, Dako) where the assay is also used toevaluate patients for antibody therapy using Herceptin®. In vivoapplications include directed cell therapy and immune system modulationand radio imaging of immune responses.

In one embodiment, an anti-OX40 antibody can be used inimmunohistochemistry of biopsy samples.

In another embodiment, an anti-OX40 antibody can be used to detectlevels of OX40, or levels of cells which contain OX40 on their membranesurface, which levels can then be linked to certain disease symptoms.Anti-OX40 antibodies described herein may carry a detectable orfunctional label. When fluorescence labels are used, currently availablemicroscopy and fluorescence-activated cell sorter analysis (FACS) orcombination of both methods procedures known in the art may be utilizedto identify and to quantitate the specific binding members. Anti-OX40antibodies described herein can carry a fluorescence label. Exemplaryfluorescence labels include, for example, reactive and conjugatedprobes, e.g., Aminocoumarin, Fluorescein and Texas red, Alexa Fluordyes, Cy dyes and DyLight dyes. An anti-OX40 antibody can carry aradioactive label, such as the isotopes ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr,⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶⁷Cu, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹¹⁷Lu, ¹²¹I, ¹²⁴I, ¹²⁵I, ¹³¹I,¹⁹⁸Au, ²¹¹At, ²¹³Bi, ²²⁵Ac and ¹⁸⁶Re. When radioactive labels are used,currently available counting procedures known in the art may be utilizedto identify and quantitate the specific binding of anti-OX40 antibody toOX40 (e.g., human OX40). In the instance where the label is an enzyme,detection may be accomplished by any of the presently utilizedcolorimetric, spectrophotometric, fluorospectrophotometric, amperometricor gasometric techniques as known in the art. This can be achieved bycontacting a sample or a control sample with an anti-OX40 antibody underconditions that allow for the formation of a complex between theantibody and OX40. Any complexes formed between the antibody and OX40are detected and compared in the sample and the control. In light of thespecific binding of the antibodies described herein for OX40, theantibodies thereof can be used to specifically detect OX40 expression onthe surface of cells. The antibodies described herein can also be usedto purify OX40 via immunoaffinity purification.

Also included herein is an assay system which may be prepared in theform of a test kit for the quantitative analysis of the extent of thepresence of, for instance, OX40 or OX40/OX40L complexes. The system ortest kit may comprise a labeled component, e.g., a labeled antibody, andone or more additional immunochemical reagents. See, e.g., Section 7.6below for more on kits.

7.6 Kits

Provided herein are kits comprising one or more antibodies describedherein or conjugates thereof. In a specific embodiment, provided hereinis a pharmaceutical pack or kit comprising one or more containers filledwith one or more of the ingredients of the pharmaceutical compositionsdescribed herein, such as one or more antibodies provided herein. Insome embodiments, the kits contain a pharmaceutical compositiondescribed herein and any prophylactic or therapeutic agent, such asthose described herein. In certain embodiments, the kits may contain a Tcell mitogen, such as, e.g., phytohaemagglutinin (PHA) and/or phorbolmyristate acetate (PMA), or a TCR complex stimulating antibody, such asan anti-CD3 antibody and anti-CD28 antibody. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Also provided herein are kits that can be used in the above methods. Inone embodiment, a kit comprises an antibody described herein, preferablya purified antibody, in one or more containers. In a specificembodiment, kits described herein contain a substantially isolated OX40antigen (e.g., human OX40) that can be used as a control. In anotherspecific embodiment, the kits described herein further comprise acontrol antibody which does not react with a OX40 antigen. In anotherspecific embodiment, kits described herein contain one or more elementsfor detecting the binding of an antibody to a OX40 antigen (e.g., theantibody can be conjugated to a detectable substrate such as afluorescent compound, an enzymatic substrate, a radioactive compound ora luminescent compound, or a second antibody which recognizes the firstantibody can be conjugated to a detectable substrate). In specificembodiments, a kit provided herein can include a recombinantly producedor chemically synthesized OX40 antigen. The OX40 antigen provided in thekit can also be attached to a solid support. In a more specificembodiment, the detecting means of the above described kit includes asolid support to which a OX40 antigen is attached. Such a kit can alsoinclude a non-attached reporter-labeled anti-human antibody oranti-mouse/rat antibody. In this embodiment, binding of the antibody tothe OX40 antigen can be detected by binding of the said reporter-labeledantibody.

The following examples are offered by way of illustration and not by wayof limitation.

8. EXAMPLES

The examples in this Section (i.e., Section 8) are offered by way ofillustration, and not by way of limitation.

8.1 Example 1: Characterization of GITR and OX40 Expression onIntratumoral Regulatory T Cells

The expression of GITR and OX40 on intratumoral regulatory T cells(Treg) and effector T cells (Teff) was characterized using flowcytometry. Briefly, cryopreserved tumor cells from multiple tumor types(ovarian, stage IIC; colorectal, stage TIM; endometrial, stage IB;renal, stage III; and non-small cell lung cancer, stage II) wereobtained from Conversant Bio, LLC. The tumor cells were isolated priorto therapeutic interventions. After being thawed, cells were treatedwith human Fc-receptor block (FcR block, Biolegend®) for 15 minutes atroom temperature to reduce non-specific binding. Samples were washedtwice. An antibody cocktail, containing antibodies recognizing CD4(BV605, OKT4, lot #B185762), CD127 (APC, A019D5, lot #B193084) and CD25(PECy7, M-A251 lot #B190207), Zombie Green™ fixable viability dye (FITC,lot #B201900) as well as anti-OX40 antibody (PE, BER-ACT35, lot#B203538), anti-GITR antibody (PE, 110416, lot #LAV0614061), or acognate isotype control antibody (PE, MOPC-21, lot #B197832) all at 2.5μg/ml, was diluted in FACS buffer (PBS, 2 mM EDTA, 0.5% BSA, pH 7.2) andadded to each sample. The samples were incubated for 30 minutes at 4° C.Prior to staining, additional samples were set aside for single staincompensation controls (CD45-FITC, CD45-PE, CD45-PECy7, CD45-APC, andCD45-BV605; all clone H130). Samples were then washed three times inFACS buffer and incubated in 1× fix-perm buffer (Foxp3 staining kit,eBioscience) for 45 minutes at room temperature in the dark. Followingfixation, cells were washed three times in 1× permeabilization buffer(Foxp3 staining kit, eBioscience) and incubated with anti-FOXP3 or ratIgG₂a antibodies at 2.5 μg/ml diluted in 1× permeabilization buffer for45 minutes at 4° C. Samples were washed two times in 1× permeabilizationbuffer, resuspended in FACS buffer, and analyzed using the LSRFortessaflow cytometer (BD Biosciences). FACS plots were analyzed using acombination of FACS DIVA and WEHI Weasel software.

Effector T cells were defined as CD4+ CD127+ CD25+/− FOXP3−. RegulatoryT cells were defined as CD4+ CD127− CD25+ FOXP3+. As shown in FIG. 1A,intratumoral regulatory T cells from endometrial cancer tumor tissue,renal cell carcinoma (RCC) tumor tissue, and non-small cell lung cancer(NSCLC) tumor tissue showed elevated expression of GITR and OX40,whereas intratumoral effector T cells from the same tumor tissues didnot.

For receptor quantification, beads with a pre-defined number of PEmolecules (Quantum™ R-PE MESF; Bangs Laboratories, Inc.) were analyzedusing defined instrument settings. Using mean fluorescence intensity(MFI) for both beads and previously analyzed GITR/OX40 antibodies, thepredicted number of GITR and OX40 receptors for intratumoral regulatoryT cells and effector T cells was calculated.

Regulatory T cells from ovarian tumor tissue, colorectal cancer (CRC)tumor tissue, endometrial cancer tumor tissue, RCC tumor tissue, andNSCLC tumor tissue all showed higher expression of GITR and OX40 thaneffector T cells from the same tumor tissues (FIG. 1B).

8.2 Example 2: Characterization of Anti-GITR/OX40 Bispecific Antibodies

In this example, anti-GITR/OX40 bispecific antibodies constructed usingGenmab DuoBody technology were examined for their binding and functionalcharacteristics. DuoBody pab1876×pab2049 and DuoBody pab1876×pab1949both comprise a GITR-binding arm (pab1876) and an OX40-binding arm(pab2049 or pab1949). Three additional DuoBody antibodies were used ascontrols: DuoBody pab1876×isotype, DuoBody pab2049×isotype and DuoBodypab1949×isotype. The SEQ ID NOs corresponding to the heavy chain andlight chain sequences of these DuoBody antibodies are listed in Table10. In addition, the bivalent monospecific antibodies pab1876, pab2049,and pab1949w were also used in some experiments. pab1876w is an IgG₁antibody comprising a heavy chain of SEQ ID NO:29 and a light chain ofSEQ ID NO:37. pab1876 comprises the same heavy and light chain sequencesas pab1876w except that it contains a T109S substitution in the lightchain constant domain (i.e., substitution of threonine with serine atposition 109 relative to the wild type light chain constant domain),numbered according to Kabat, which facilitates the cloning of thevariable region in frame to the constant region. This mutation is aconservative modification that does not affect antibody binding orfunction. pab2049w is an IgG₁ antibody comprising a heavy chain of SEQID NO:59 and a light chain of SEQ ID NO:67. pab2049 comprises the sameheavy and light chain sequences as pab2049w except for a T109S mutationin the light chain constant region, numbered according to Kabat.pab1949w is an IgG₁ antibody comprising a heavy chain of SEQ ID NO:59and a light chain of SEQ ID NO:69. pab1949 comprises the same heavy andlight chain sequences as pab1949w except for a T109S mutation in thelight chain constant region, numbered according to Kabat.

TABLE 10 DuoBody heavy chain (HC) and light chain (LC) sequences Firstarm Second arm HC LC HC LC (SEQ (SEQ (SEQ (SEQ DuoBody ID NO:) ID NO:)ID NO:) ID NO:) pab1876 × pab2049 31 38 61 68 pab1876 × pab1949 31 38 6170 pab1876 × isotype 31 38 N/A N/A pab2049 × isotype 61 68 N/A N/Apab1949 × isotype 61 70 N/A N/A pab1876 × pab2049 without 76 38 120 68heavy chain terminal lysine pab1876 × pab1949 without 76 38 120 70 heavychain terminal lysine pab1876 × isotype without 76 38 N/A N/A heavychain terminal lysine pab2049 × isotype without 120 68 N/A N/A heavychain terminal lysine pab1949 × isotype without 120 70 N/A N/A heavychain terminal lysine

8.2.1 Selectivity of Anti-GITR/OX40 Bispecific Antibody

The selectivity of DuoBody pab1876×pab2049 for GITR and OX40 wasassessed against other members of the TNFR superfamily using suspensionarray technology. A number of recombinant proteins of the TNFRsuperfamily, including recombinant human GITR-His (Sino Biological, 50μg/ml), recombinant human OX40-His (Sino Biological, 50 μg/ml),recombinant human lymphotoxin beta receptor (LTBR)-Fc (AcroBiosystems,65 μg/ml), recombinant human death receptor 6 (DR6)-bio (SinoBiological, 50 μg/ml), recombinant human tumor necrosis factor-like weakinducer of apoptosis receptor (TWEAKR)-Fc (Sino Biological, 50 μg/ml),recombinant human CD137-Fc (SrtA-bio, 50 μg/ml), and recombinant humanB-cell activating factor receptor (BAFFR)-Fc (R&D Systems, 50 μg/ml),were coupled to Luminex beads using goat anti-human IgG F(ab′)₂ (JacksonImmuno Research, COOH coupling, 100 μg/ml, pH 5.0). DuoBodypab1876×pab2049 was then incubated at multiple concentrations (8333,833.3, 83.3 and 8.33 ng/ml final) with the antigen-coupled beads for 1hour at 20° C. (650 RPM in the dark). Following washing to removenon-specific binding (two times in PBS), the beads were incubated withdetection antibody (phycoerythrin-coated goat-anti-huIgG F(ab′) ₂, 2.5μg/ml final) for 1 hour at 20° C. (650 RPM in the dark). Beads were thenwashed two times and read on a Luminex 200®. Binding above (+) or below(−) threshold was determined by cutoff detection values (based on LX200controls).

DuoBody pab1876×pab2049 showed specific binding to human GITR and OX40,and no significant binding to other TNFR family members was observed attested concentrations (Table 11).

TABLE 11 Selectivity of DuoBody pab1876 × pab2049 to TNFR superfamilymembers Target GITR OX40 LTBR DR6 TWEAKR CD137 BAFFR Binding + + − − − −−

8.2.2 Binding of Anti-GITR/OX40 Bispecific Antibody to Cells ExpressingGITR and OX40

The binding of DuoBody pab1876×pab2049 to cells co-expressing GITR andOX40, cells expressing only GITR, and cells expressing only OX40 wasexamined by flow cytometry.

Hut102 cells (human T cell lymphoma, ATCC) were incubated for 72 hoursin RPMI media, supplemented with 1 μg/ml phytohaemagglutinin (PHA) and10% heat-inactivated FBS, at 37° C. and 5% CO₂ to induce GITR and OX40expression. Cells ectopically expressing GITR or OX40 were generated bytransduction of lentiviral vectors (EF1a promoter) into Jurkat cells.Stable clones were generated via single-cell sorting (FACS ARIA Fusion).Expression was verified by flow cytometry. For binding analysis, stableJurkat cells or activated Hut102 cells were incubated with testantibodies (12-point dose titration, 0.01-10,000 ng/ml) diluted in FACSbuffer (PBS, 2 mM EDTA, 0.5% BSA, pH 7.2) for 30 minutes at 4° C.Samples were washed two times in FACS buffer and then incubated withAPC-conjugated mouse anti-human kappa detection antibody (LifeTechnologies, HP6062) for 30 minutes at 4° C. Samples were then washedtwo times and analyzed using the LSRFortessa flow cytometer (BDBiosciences). FACS plots were analyzed using a combination of FACS DIVAand WEHI Weasel software. Data were plotted with Graphpad Prismsoftware.

As shown in FIG. 2A, DuoBody pab1876×pab2049 showed enhanced binding tocells co-expressing GITR and OX40, as compared to the bivalentmonospecific antibodies pab1876 and pab2049. The enhanced binding tocells co-expressing GITR and OX40 was contributed to by both arms, asreplacing either arm with an isotype arm in the two control DuoBodyantibodies, pab1876×isotype and pab2049× isotype, led to reduced bindingto activated Hut102 cells. As expected, for cells that only expressedGITR or OX40 but not both, DuoBody pab1876×pab2049 bound more weaklythan bivalent monospecific antibodies pab1876 and pab2049 did at all butthe highest concentrations tested (FIGS. 2B and 2C).

8.2.3 Effect of Anti-GITR/OX40 Bispecific Antibody on FcγRIIIA ReporterCell Line

Next, the ability of DuoBody pab1876×pab2049 to engage GITR and OX40 andsignal via FcγRIIIA was evaluated using a reporter cell line expressingFcγRIIIA (Promega) together with target cells co-expressing GITR andOX40. Engineered Jurkat cells stably expressing the FcγRIIIA V158variant and an NFAT response element driving expression of fireflyluciferase were used as effector cells. Binding of the antibody/antigencomplex, wherein the antigen is located on the surface of the targetcells, to FcγRIIIA signals to the promoter/reporter construct of theeffector cells and results in luciferase gene transcription.

Natural regulatory T cells (nTreg) were activated to generate targetcells co-expressing GITR and OX40. PBMCs were isolated from healthydonor buffy coats via Ficoll gradient (Research Blood Components, LLC)and subjected to magnetic-based nTreg enrichment (Mitenyi Biotec,130-093-631, lot 5150629039). Cells were then activated using MiltenyiBiotec's Treg Expansion Kit (130-095-345, lot 5150420196) for 8 days inRPMI media, supplemented with 10% heat-inactivated FBS, at 37° C. and 5%CO₂. Fresh media containing the Treg Expansion Kit was added to theisolated nTregs every 3-4 days. On day 8, cell expression of GITR andOX40 was confirmed by flow cytometry. Briefly, 20,000 cells were treatedwith human Fc-receptor block for 15 minutes at room temperature toreduce non-specific binding (FcR block, Biolegend). Samples were washedtwice and an antibody cocktail, containing antibodies recognizing CD4(BV605, OKT4, lot B185762), CD127 (APC, A019D5, lot B193084), and CD25(PECy7, M-A251, lot B195168) and Zombie Green™ fixable viability dye(FITC, lot B201900), as well as anti-OX40 antibody (PE, BER-ACT35, lot#B203538), anti-GITR antibody (PE, 110416, lot #LAV0614061), or acognate isotype control antibody (PE, MOPC-21, lot #B197832) all at 2.5μg/ml, was diluted in FACS buffer (PBS, 2 mM EDTA, 0.5% BSA, pH 7.2),added to each sample and incubated for 30 minutes at 4° C. Prior tostaining, additional samples were set aside for single staincompensation controls (CD45-FITC, CD45-PE, CD45-PECy7, CD45-APC, andCD45-BV605; all clone H130). Samples were then washed three times inFACS buffer and incubated in 1× fix-perm buffer (Foxp3 staining kit,eBioscience, lot E00029-1691) for 45 minutes at room temperature in thedark. Following fixation, cells were washed three times in 1×permeabilization buffer (Foxp3 staining kit, eBioscience) and incubatedwith anti-FOXP3 (eFluor450, PCH101, lot E11056-1635) or rat IgG2a(eFluor450, eBR2a, lot E08519-1633) antibodies at 2.5 μg/ml diluted in1× permeabilization buffer for 45 minutes at 4° C. Samples were washedtwo times in 1× permeabilization buffer, resuspended in FACS buffer andanalyzed using the LSRFortessa flow cytometer (BD Biosciences).

As shown in FIG. 3A, activated nTregs expressed both GITR and OX40 onthe cell surface.

To assess impact on FcγRIIIA reporter cells, 125,000 nTregs, activatedfor 8 days, were incubated with increasing concentrations (6-point dosetitration, 0.04 to 10 μg/ml) of DuoBody pab1876×pab2049, the bivalentmonospecific antibody pab1876, the bivalent monospecific antibodypab2049, or an isotype control antibody. FcγRIIIA^(V158)-expressing NFATreporter cells were added to the antibody-nTreg mixture at a 1:1 ratio(125,000 cells) in RPMI media, supplemented with 4% heat-inactivatedlow-IgG FBS, at 37° C. and 5% CO₂. After a 20-hour incubation, Bio-Glo™Luciferase Assay Substrate (Promega, G720A) was added to each sample(1:1 v/v). Luminescence was measured using the EnVision® MultilabelPlate Reader (Perkin-Elmer). FACS plots were analyzed using acombination of FACS DIVA and WEHI Weasel software. Data were plottedwith Graphpad Prism software.

Consistent with the observation that DuoBody pab1876×pab2049 showedenhanced binding to cells co-expressing GITR and OX40, as compared tothe bivalent monospecific antibodies pab1876 and pab2049 (FIG. 2A), whenbound to GITR- and OX40-co-expressing nTregs, DuoBody pab1876×pab2049demonstrated a stronger activation of FcγRIIIA than pab1876 and pab2049did (FIG. 3B).

Furthermore, the ability of DuoBody pab1876×pab2049 to engage GITR orOX40 and signal via FcγRIIIA was evaluated using the FcγRIIIA-expressingreporter cell line described above together with target cells expressingGITR or OX40 but not both. Briefly, Jurkat target cells expressing GITRor OX40 were counted and resuspended at a concentration of 6×10⁶cells/ml in RPMI-1640 with 4% low-IgG FBS. To the inner 60 wells ofmultiple 96-well white assay plates, 11.1 of the cell suspension wasadded to each well. Test antibodies were serially diluted with 3-folddilutions with a starting final concentration of 10 μg/ml. In duplicatewells, 25 μl of each antibody dilution was added to the target cells.Finally, FcγRIIIA^(V158)-expressing NFAT reporter cells were resuspendedat a concentration of 6×10⁶ cells/ml in RPMI-1640 with 4% low-IgG FBS.25 μl of these reporter cells were added to each well resulting in a 1:1effector to target ratio. Plates were incubated for 20 hours at 37° C.and 5% CO₂. After this incubation, Bio-Glo Luciferase Assay Reagent(Promega) was thawed at room temperature and 75 μl was added to eachwell of the 96-well white assay plates. Within 5-10 minutes,luminescence was measured using the EnVision multilabel plate reader(PerkinElmer). Background luminescence (blank outer wells) wassubtracted from each sample reading and the adjusted relative lightunits (RLU) were recorded. Data were plotted with Graphpad Prismsoftware. The antibodies tested were: DuoBody pab1876×pab2049, thebivalent anti-GITR antibody pab1876 (F405L/F405L), the bivalentanti-OX40 antibody pab2049 (K409R/K409R), DuoBody pab1876×isotype,DuoBody pab2049× isotype, and an isotype control antibody. The antibodypab1876 (F405L/F405L) comprises a F405L substitution in both heavy chainconstant regions and the antibody pab2049 (K409R/K409R) comprises aK409R substitution in both heavy chain constant regions, numberedaccording to the EU numbering system.

For cells that only expressed GITR or OX40 but not both, consistent withthe observation that DuoBody pab1876×pab2049 bound more weakly thanpab1876 and pab2049 did (FIGS. 2B and 2C), when bound to Jurkat cellsexpressing GITR or OX40 but not both, DuoBody pab1876×pab2049demonstrated a weaker activation of FcγRIIIA than pab1876 (F405L/F405L)and pab2049 (K409R/K409R) did at all but the highest concentrationstested (FIGS. 3C and 3D). 8.2.4 Effect of anti-GITR/OX40 bispecificantibody on NK cell-mediated ADCC activity

In this example, the ability of DuoBody pab1876×pab2049 to inducenatural killer (NK) cell-mediated antibody-dependent cellularcytotoxicity (ADCC) towards cells co-expressing GITR and OX40 wasexamined.

Human PBMCs isolated via ficoll gradient from healthy donor buffy coats(Research Blood Components, LLC) were further enriched for effector Tcells or natural Tregs using magnetic bead isolation (MACS, Miltenyi,130-094-775). The enriched effector T cells or Tregs were activated withCD3-CD28 microbeads (1:1 beads:cells, Invitrogen, 11132D) andrecombinant human IL-2 (20 U/ml for effector T cells; 100 U/ml forTregs) (Peprotech, 200-02) for 7 days in RPMI media, supplemented with10% heat-inactivated FBS at 37° C. and 5% CO₂. Following stimulation,the cells were evaluated for GITR and OX40 expression via flowcytometry. To reduce non-specific binding, the cells were incubated withan FcγR blocking antibody (Biolegend, 422302) for 15 minutes at ambienttemperature. The samples were then washed twice and incubated with alineage antibody panel of CD3, CD4, CD8, and CD25 as well as a fixablelive/dead marker for 30 minutes at 4° C. For Treg delineation, thesamples were then washed twice, fixed, permeabilized, and incubated withan anti-FOXP3 antibody (eBiosciences, clone #PCH101) for 30 minutes at4° C. The samples were then washed twice and analyzed using theLSRFortessa flow cytometer (BD Biosciences). The flow cytometry plotswere analyzed using a combination of FACS DIVA and WEHI Weasel software.To evaluate ADCC activities, primary NK cells were isolated from healthydonor PBMCs via magnetic bead separation (MACS, Miltenyi, 130-092-657).The NK cells were rested overnight with 20 U/ml of recombinant humanIL-2 (Peprotech, 200-02). The NK cells were co-cultured with targetcells (effector T cells or Tregs) and incubated with antibodies(titration range: 0.0004-1.9 μg/ml) for four hours at an E:T ratio of10:1 in RPMI 1640 phenol red-free medium supplemented withheat-inactivated 0.5% FBS. There were five treatment groups: isotypecontrol, pab1876 alone, pab2049 alone, DuoBody pab1876×pab2049, and acombination of pab1876 and pab2049. In the last group, pab1876 andpab2049 were added at equimolar concentrations to achieve a finalconcentration same as that of other groups. A total of 2×10⁵ targetcells (effector T cell or Treg) and 2×10⁶ NK cells were added in eachwell in a total volume of 100 μl. Following incubation, cell lysis, asevidenced by lactate dehydrogenase (LDH) release, was measured using theCytoTox 96 non-radioactive cytotoxicity assay (Promega, G1780) accordingto the manufacturer's instructions. Cytotoxicity (% cell lysis) wasdetermined using the following formula: %Cytotoxicity=(Experimental−Effector Spontaneous−TargetSpontaneous)/(Target Maximum−Target Spontaneous)*100.

As shown in FIG. 3E, activated Tregs expressed higher levels of GITR andOX40 than activated effector T cells. Consistent with this differentialexpression pattern, the antibodies against GITR and/or OX40 did notinduce significant lysis of activated effector T cells above backgroundlevels (FIG. 3F), whereas the same antibodies induced strong NKcell-mediated ADCC activities towards activated Tregs in a dosedependent manner (FIG. 3G). Notably, DuoBody pab1876×pab2049 inducedhigher levels of lysis of activated Tregs than pab1876 alone, pab2049alone, or a combination of pab1876 and pab2049.

8.2.5 Effect of Anti-GITR/OX40 Bispecific Antibody on Human T CellsFollowing Staphylococcus Enterotoxin A (SEA) Stimulation

The functional activity of DuoBody pab1876×pab2049 on primary human Tcells was assessed following Staphylococcus Enterotoxin A (SEA)stimulation. Briefly, PBMCs isolated via Ficoll gradient from healthydonor buffy coats (Research Blood Components, LLC) were incubated inRPMI media, supplemented with 100 ng/ml SEA superantigen (Sigma-Aldrich)and 10% heat-inactivated FBS, together with increasing concentrations oftest antibodies (7-point dose titration, 0.02-20 μg/ml) for 5 days at37° C. and 5% CO₂. Following incubation, cell-free supernatant wasassayed for IL-2 production using an AlphaLISA immunoassay(Perkin-Elmer). Data were collected using the EnVision ° MultilabelPlate Reader (Perkin-Elmer) and the concentration of IL-2 was determinedusing an IL-2 standard curve. Values were interpolated and plotted usingGraphpad Prism software.

DuoBody pab1876×pab2049 induced IL-2 production in this primary humanPBMC assay using cells from two donors (FIGS. 4A and 4B). Importantly,DuoBody pab1876×pab2049 was able to induce high levels of IL-2production at pharmacologically relevant antibody concentrations. IL-2production induced by DuoBody pab1876×pab2049 is a substantiallyincreasing function of antibody concentration across a wide range ofantibody concentrations (e.g., between 0.08 and 20 μg/ml in FIG. 4A andbetween 0.009 and 20 μg/ml in FIG. 4B).

8.3 Example 3: Anti-GITR/OX40 Bispecific Antibodies as AntagonistAntibodies

The activation of GITR and OX40 signaling depends on receptor clusteringto form higher order receptor complexes that efficiently recruit apicaladapter proteins to drive intracellular signal transduction. Withoutbeing bound by theory, one possible mechanism for the agonistic activityof DuoBody pab1876×pab2049 shown in Section 8.2.4 is by clustering GITRand/or OX40 receptors through Fc-Fc receptor (FcR) co-engagement onaccessory myeloid or lymphoid cells, e.g., dendritic cells, monocytes,macrophages, natural killer (NK) cells, and/or B cells. Some tumor cellsexpressing FcRs may also mediate antibody clustering, e.g., hematologiccancers (acute myelogenous leukemia (AML), plasma cell cancers andnon-Hodgkin's lymphoma (NHL)) as well as certain solid (epithelial)tumor cells (e.g. melanoma). Consequently, one approach for developingan anti-GITR/OX40 bispecific antagonist antibody is to select abispecific antibody that competes with GITR ligand (GITRL) and OX40ligand (OX40L) for binding to their respective receptors, and diminishor eliminate the binding of the Fc region of the bispecific antibody toFc receptors. In this example, two reporter assays were developed tofirst, confirm the loss of the agonistic activity of DuoBodypab1876×pab2049 in the absence of FcR interaction, and second, examinethe ability of DuoBody pab1876×pab2049 to antagonize GITRL- andOX40L-induced signaling through GITR and OX40 receptors.

8.3.1 Effect of Anti-GITR/OX40 Bispecific Antibody on GITRNF-κB-Luciferase Reporter Cells

First, DuoBody pab1876×pab2049 was evaluated for its agonistic activityon GITR using a GITR reporter assay. This reporter assay was built usingJurkat cells which expressed minimum amount, if any, of FcR, diminishingthe possibility of FcR-mediated clustering of the GITR molecules.

Cells ectopically expressing GITR as well as NF-κB-luciferase (Nanoluciferase, NanoLuc®) reporter were generated by transduction oflentiviral vectors (EF1a promoter) into Jurkat cells. Stable clones weregenerated via single-cell sorting (FACS ARIA Fusion). Expression of GITRwas verified by flow cytometry. To evaluate agonistic activity,Jurkat-huGITR-NF-κB-luciferase cells were incubated with increasingconcentrations of DuoBody pab1876×pab2049 or trimeric GITRL (12-pointdose titration, 0.05-10,000 ng/ml) for 2 hours in RPMI media,supplemented with 10% heat-inactivated FBS, at 37° C. and 5% CO₂. Fordetection of luciferase activity, samples were incubated with preparedNanoGlo® Luciferase Assay Substrate (Promega, 1:1 v/v) in passive lysisbuffer for 5 minutes at room temperature. Data were collected using theEnVision® Multilabel Plate Reader (Perkin-Elmer). Values were plottedusing Graphpad Prism software.

In contrast to trimeric GITRL, which induced high levels ofNF-κB-luciferase activity as represented by RLU (relative luciferaseunits), DuoBody pab1876×pab2049 showed minimal agonistic activity of theGITR reporter cells even at the highest concentration tested (FIG. 5A).

Next, the ability of DuoBody pab1876×pab2049 to neutralize GITRL-inducedNF-κB signaling was examined as a surrogate readout of the DuoBody'sligand blocking activity.

Briefly, Jurkat-huGITR-NF-κB-luciferase cells were incubated withincreasing concentrations of DuoBody pab1876×pab2049 or an isotypecontrol antibody (10-point dose titration, 0.5-10,000 ng/ml) for 30minutes. Samples were then washed two times with RPMI, resuspended in 1μg/ml of trimeric GITRL and incubated for additional 2 hours at 37° C.Luciferase activity was detected and analyzed as described above. Todetermine % GITRL activity, the RLU value for GITRL (1 μg/ml) withoutaddition of antibody was established as 100% activity. Relative valuesfor DuoBody pab1876×pab2049 and the isotype control were calculatedaccordingly.

As shown in FIG. 5B, pre-incubation of Jurkat-huGITR-NF-κB-luciferasereporter cells with increasing concentrations of DuoBody pab1876×pab2049significantly reduced GITRL-induced NF-κB-luciferase activity in adose-dependent manner.

8.3.2 Effect of Anti-GITR/OX40 Bispecific Antibody on OX40NF-κB-Luciferase Reporter Cells

Similarly, an OX40 reporter assay was developed to test the agonisticactivity of DuoBody pab1876×pab2049 on OX40-expressing cells. This OX40reporter assay was also constructed using Jurkat cells where FcRexpression was minimal.

Cells ectopically expressing OX40 as well as NF-κB-luciferase (Nanoluciferase, NanoLuc®) reporter were generated by transduction oflentiviral vectors (EF1a promoter) into Jurkat cells. Stable clones weregenerated via single-cell sorting (FACS ARIA Fusion). Expression of OX40was verified by flow cytometry. To evaluate agonistic activity,Jurkat-huOX40-NF-κB-luciferase cells were incubated with increasingconcentrations of multimeric OX40L, DuoBody pab1876×pab2049 or anisotype control antibody (10-point dose titration, 0.5-10,000 ng/ml) for2 hours in RPMI media, supplemented with 10% heat-inactivated FBS, at37° C. and 5% CO₂. For detection of luciferase activity, samples wereincubated with prepared Nano-Gb® Luciferase Assay Substrate (Promega,1:1 v/v) in passive lysis buffer for 5 minutes at room temperature. Datawere collected using the EnVision® Multilabel Plate Reader(Perkin-Elmer). Values were plotted using Graphpad Prism software.

While multimeric OX40L induced NF-κB-luciferase activity over a widerange of concentrations, minimal luciferase signal was observed afterincubation with DuoBody pab1876×pab2049 (FIG. 6A).

Next, DuoBody pab1876×pab2049 was assessed for its ability to blockOX40L-induced NF-κB signaling. Jurkat-huOX40-NF-κB-luciferase cells wereincubated with increasing concentrations of DuoBody pab1876×pab2049 oran isotype control antibody (10-point dose titration, 0.5-10,000 ng/ml)for 30 minutes. Samples were then washed two times with RPMI,resuspended in 1 μg/ml of multimeric OX40L and incubated for additional2 hours at 37° C. Luciferase activity was detected and analyzed asdescribed above. To determine % OX40L activity, the RLU value for OX40L(1 μg/ml) without addition of antibody was established as 100% activity.Relative values for DuoBody pab1876×pab2049 and the isotype control werecalculated accordingly.

As shown in FIG. 6B, pre-incubation of Jurkat-huOX40-NF-κB-luciferasereporter cells with increasing concentrations of DuoBody pab1876×pab2049significantly reduced OX40L-induced NF-κB-luciferase activity in adose-dependent manner.

8.4 Example 4: Epitope Mapping of Anti-GITR Antibodies

This example characterizes the binding epitope of the followinganti-GITR antibodies: a chimeric parental 231-32-15 antibody and itshumanized versions (pab1876, pab1875, pab1967, pab1975, and pab1979). Inaddition, a reference anti-GITR antibody named m6C8 was also used insome studies for comparison. The antibody m6C8 was generated based onthe variable regions of the antibody 6C8 provided in PCT ApplicationPub. No. WO 2006/105021 (herein incorporated by reference). The SEQ IDNOs corresponding to the heavy chain variable regions and light chainvariable regions of these anti-GITR antibodies are listed in Table 12.

TABLE 12 VH and VL sequences of anti-GITR antibodies Antibody VH (SEQ IDNO:) VL (SEQ ID NO:) 231-32-15 101 102 pab1876 18 19 pab1875 18 103pab1967 20 21 pab1975 22 23 pab1979 24 23 m6C8 104 105

8.4.1 Epitope Competition—Cell Binding Assay

To confirm that the humanized variant antibodies retained the epitopespecificity of the chimeric 231-32-15 parental antibody, a cell bindingassay was performed. 1624-5 pre-B cells expressing the chimeric parental231-32-15 antibody were harvested and 1×10⁶ cells were resuspended in200 μl FACS buffer plus: i) biotinylated GITR (GITR-bio) (1:1000),preincubated for 15 min with 2 μg chimeric parental 231-32-15 antibody;ii) GITR-bio (1:1000), preincubated for 15 min with 2 μg pab1875; iii)GITR-bio (1:1000), preincubated for 15 min with 2 μg pab1876; or iv)GITR-bio (1:1000). The cells were incubated for 20 min at 4° C. and thenwashed with 4 ml FACS buffer and centrifuged for 5 min at 300 g at 4° C.The cell pellet was resuspended in 200 μl FACS buffer plusstreptavidin-PE (1:1000) and then incubated and washed as before. Thecells were then resuspended in 20011.1FACS buffer for analysis using aFACS-AriaII (BD Biosciences).

FIG. 7 shows that the humanized variant antibodies retained the epitopespecificity of the chimeric parental 231-32-15 antibody. The right-handprofile shows the binding of GITR-bio to 1624-5 pre-B cells expressingthe chimeric parental 231-32-25 antibody. However, when GITR-bio waspre-incubated with either chimeric parental 231-32-15, pab1875 orpab1876 antibodies, there was a loss of binding of GITR-bio to the1624-5 cells (left-hand profile). The overlapping FACS profiles indicatethat the humanized variants also show very similar GITR bindingproperties to each other and to the chimeric parental 231-32-15antibody.

8.4.2 Epitope Competition—Suspension Array Technology

Anti-GITR antibodies (25 μl) were diluted to 2 μg/ml in assay buffer(Roche 11112589001) and incubated with 1500 Luminex® beads (5 μl,Luminex Corp, no 5 LC10005-01) coupled with anti-human IgG(F(ab)₂-specific, JIR, 105-006-097 overnight in 0.5 ml LoBind tubes(Eppendorf, 0030108.116) under shaking conditions, in the dark. Thismixture was then transferred to pre-wetted 96-well filter plates(Millipore, MABVN1250). Plates were washed twice with 20011.1/well PBSto remove unbound antibody. At the same time 20 μg/ml of either the sameanti-GITR antibodies, different anti-GITR antibodies, or assay bufferwere incubated with 20 μl (1 μg/ml) R-PE labeled GITR antigen (R&Dsystems, di-sulfide-linked homodimer; 689-GR; in-house labeled withAbDSerotec LYNX Kit, LNK022RPE) for 1 hour in the dark at 650 rpm. Thebead mixture and the antigen/antibody mixture were mixed 1:1 (20 μl fromeach) and incubated for one additional hour under shaking conditions(20° C., 650 rpm). Directly before the measurement, 40 μl of assaybuffer was added to each well and analysis was performed using aLuminex® 200 system (Millipore) and a readout of 100 beads in 48 μlsample volume. Binding was determined using the MFI values of thenon-competed control (100% binding, only assay buffer as competingcompound).

When the chimeric parental 231-32-15 antibody was used as the capturedantibody, full binding competition was observed with both humanizedantibodies pab1875 and pab1876. When the anti-GITR antibody m6C8 wasused as the captured antibody, no competition of binding was observedwith the chimeric parental 231-32-15 antibody or the two humanizedvariants pab1875 and pab1876 (data not shown). These results indicatethat m6C8 and the anti-GITR antibodies described herein recognizedifferent epitopes on human GITR.

8.4.3 Epitope Competition—Surface Plasmon Resonance

For epitope binning using surface plasmon resonance the “in tandemapproach” was used (Abdiche Y N et al., (2009) Analytical Biochemistry,386: 172-180). For that purpose different chip surfaces were generatedon a CM5 sensor chip (GE Healthcare, Series S CM5, BR-1005-30) usingimmobilization of different densities of GITR antigen (R&D systems,disulfide-linked homodimer; 689-GR). Flow cell 2 contained GITR antigenin low density (667 RU), medium density was assessed in flow cell 3(1595 RU) and in flow cell 4, high density was achieved (4371 RU). Inflow cell 1, ovalbumin (1289 RU, Pierce ThermoFisher 77120) wasimmobilized for reference. Immobilization was performed according to astandard protocol from the manufacturer (GE Healthcare) for aminecoupling (activation of surface with 0.4 M EDC and 0.1 M NHS, GEHealthcare Amine coupling kit, BR-1000-50). Unreacted groups wereinactivated with 1 M ethanol-amine-HCl, pH8.5. Afterwards anti-GITRantibodies were run through the different surfaces at a concentration of300 nM (45 μg/ml) for 240 seconds at 5 μl/min. Using these conditionssaturation of the GITR surface should have been reached. A dissociationtime of 60 seconds was included before adding the competing antibody(300 nM, 5 μl/min). Regeneration of the chip surface was performed using10 mM Glycine pH2.0 (GE Healthcare, BR-1003-55) for 60 seconds at 10μl/min. Binning was performed using the response units (RU) of thenon-competed control (100% binding, saturating conditions).

As is shown in FIG. 8 , when the chimeric parental 231-32-15 antibody isfirst bound to GITR, no further binding of this antibody occurs.However, when the chimeric parental 231-32-15 antibody is first bound toGITR and the antibody m6C8 is applied, this antibody is still able tobind to GITR.

8.4.4 Epitope Mapping—PCR Mutagenesis and Alanine Scanning

In order to map the epitope on GITR to which anti-GITR antibodiesdescribed herein bind, error prone PCR was used to generate variants ofthe human GITR antigen. The variant GITR proteins were expressed on thesurface of cells in a cellular library and these cells were screened forbinding of the anti-GITR antibodies. As a positive control, a polyclonalanti-GITR antibody was used to confirm proper folding of the GITRprotein. For variants of the human GITR antigen to which reduced or noantibody binding occurred, alanine scanning mutagenesis was performed todetermine the precise epitope residues that were required for binding bythe anti-GITR antibodies described herein.

8.4.4.1 Generation of Human GITR Variants

Error prone PCR mutagenesis was used to generate variants of human GITRwith random mutations in the extracellular domain. For error prone PCR,the GeneMorphII Random Mutagenesis Kit (Stratagene) was used, accordingto the manufacturer's instructions. In brief, 20 PCR cycles in a volumeof 50 μl was performed using an in-house construct as template (13 ng,construct number 4377 pMA-T-huGITR), 0.05 U/μl Mutazyme II DNApolymerase, 1× Mutazyme II reaction buffer, 0.2 μM of each primer and0.2 mM of each deoxynucleoside-triphosphate (dATP, dCTP, dGTP, anddTTP). The samples were amplified by PCR (Eppendorf, Germany) using thefollowing program: 95° C. for 2 min; 20 cycles of 95° C. for 30 sec, 56°C. for 30 sec, 72° C. for 1 min; and a final extension step of 72° C.for 10 min. The PCR product was gel purified using 1% agarose gel, theDNA band corresponding to the expected size of 720 bp was cut out andgel extraction was done using a NucleoSpin Gel and PCR cleanup kit fromMacherey&Nagel according to the product manual. Purified DNA was ligatedinto an in-house expression vector via XhoI/EcoRI sites using T4 DNAligase and a ratio of 1:3 (vector:insert). Ligation (25° C.) was stoppedafter 2 hours with a heat denaturation step for 10 min at 65° C. DNAfrom the ligation reaction was EtOH precipitated using yeast t-RNA.Standard digestion and ligation techniques were used. The ligationreaction was electroporated into DH10B cells (E. coli ElectroMax DH10Belectrocompetent cells, Invitrogen; 1900V/5 ms). Electroporated bacteriawere plated onto LB-agar+100 μg/ml ampicillin plates and approximately1.9×10⁸ colonies were obtained.

All electroporated bacteria were then scratched from the plates and usedfor large-scale DNA plasmid preparation (Macherey&Nagel, NucleoBond XtraMaxi Plus Kit), according to the manufacturer's instructions to generatea DNA library. A restriction enzyme digestion with XhoI/EcoRI andBsrGI/EcoRI was performed to quality control the library. Single cloneswere picked and sent for sequencing to determine the final librarydiversity.

8.4.4.2 Generation of a Cellular Library with Human GITR Variants

Standard techniques of transfection followed by transduction were usedto express human GITR mutants on the surface of 1624-5 cells. For thegeneration of retroviral particles, a DNA library and vectors expressingretroviral proteins Gag, Pol and Env were transfected into a retroviralpackaging cell line (HEK cells) using X-tremeGENE 9 DNA transfectionreagent (Roche Diagnostics GmbH, Germany). The resulting retroviralparticles accumulated in the cell culture supernatant of the retroviralpackaging cells. Two days post transfection cell-free viral vectorparticle-containing supernatants were harvested and subjected tospin-infection of 1624-5 cells. A transduction efficiency (% human GITRexpressing cells) of roughly 4% was obtained. Upon continuous culturefor at least one additional day, cells were selected using puromycin(1.5 μg/ml). Untransduced cells served as negative controls (NC). Afterantibiotic selection, most cells stably expressed the human GITR antigenlibrary on the cell surface. Non-viable cells were removed via a Ficollseparation step.

FACS was used to select cells expressing correctly folded human GITRmutants using a polyclonal anti-GITR antibody and to subsequently selectindividual cells expressing human GITR variants that did not bind to theanti-GITR chimeric parental 231-32-15 antibody. In brief, antibodybinding cells were analyzed by FACS and cells that exhibited specificantibody binding were separated from the non-binding cell population bypreparative, high-speed FACS (FACSAriaII, BD Biosciences). Antibodyreactive or non-reactive cell pools were expanded again in tissueculture and, due to the stable expression phenotype of retrovirallytransduced cells, cycles of antibody-directed cell sorting and tissueculture expansion were repeated, up to the point that a clearlydetectable anti-GITR antibody (chimeric parental 231-32-15) non-reactivecell population was obtained. This anti-GITR antibody (chimeric parental231-32-15) non-reactive cell population was subjected to a final,single-cell sorting step. After several days of cell expansion, singlecell sorted cells were again tested for non-binding to anti-GITRchimeric parental 231-32-15 antibody and binding to a polyclonalanti-GITR antibody using 96 well plate analysis on a FACSCalibur (BDBiosciences).

8.4.4.3 Epitope Analysis

To connect phenotype (polyclonal anti-GITR+, chimeric parental231-32-15-) with genotype, sequencing of single cell sorted huGITRvariants was performed. FIGS. 9A and 9B show the alignment of sequencesfrom these variants. The amino acid residues in FIGS. 9A and 9B arenumbered according to the immature amino acid sequence of human GITR(SEQ ID NO:41). Sequencing identified regions with increased mutationsor “hot spots” (e.g., P62 and G63), providing an indication of theepitope on human GITR recognized by anti-GITR chimeric parental231-32-15 antibody.

To confirm the precise amino acids of human GITR involved in binding toanti-GITR antibodies, alanine replacement of hot spot amino acids wasperformed. The following positions (numbered according to SEQ ID NO:41)were separately mutated to an Alanine: P28A, T29A, G30A, G31A, P32A,T54A, T55A, R56A, C57A, C58A, R59A, D60A, Y61A, P62A, G63A, E64A, E65A,C66A, C67A, S68A, E69A, W70A, D71A, C72A, M73A, C74A, V75A, and Q76A.Standard techniques of transfection followed by transduction were usedto express these human GITR alanine mutants on the surface of 1624-5cells.

Finally, alanine mutants expressed on 1624-5 cells were tested in flowcytometry (FACSCalibur; BD Biosciences) for the binding of the anti-GITRhumanized antibodies pab1876, pab1967, pab1975 and pab1979, and thereference antibody m6C8. Briefly, 1624-5 cells expressing individualhuman GITR alanine mutants were incubated with 2 μg/ml of the monoclonalanti-GITR antibody pab1876, pab1967, pab1975, pab1979, or m6C8; or apolyclonal anti-GITR antibody (AF689, R&D systems) conjugated with APC,and Fc receptor block (1:200; BD Cat no. 553142) diluted in 100 μl FACSbuffer (PBS+2% FCS) for 20 min at 4° C. After washing, the cells wereincubated with a secondary anti-IgG antibody if necessary for detection(APC conjugated; BD Cat no. 109-136-097) diluted in 100 μl FACS buffer(PBS+2% FCS) for 20 min at 4° C. The cells were then washed and acquiredusing a flow cytometer (BD Biosciences). The mean fluorescence intensity(MFI) value of the tested monoclonal antibody was divided by the MFIvalue of the polyclonal antibody, generating an MFI ratio (monoclonalantibody/polyclonal antibody) for individual GITR alanine mutants. Anaverage MFI ratio (“AMFI ratio”) was calculated based on the individualMFI ratios for all the mutants. FIG. 10A is a table summarizing thebinding of pab1876, pab1967, pab1975, pab1979 and the reference antibodym6C8 to 1624-5 cells expressing human GITR alanine mutants. Anindividual MFI ratio that is above 60% of the AMFI ratio is consideredto indicate similar binding, after normalization, of that of thepolyclonal antibody and is represented by “+” in FIG. 10A. An individualMFI ratio that is between 30% and 60% of the AMFI ratio is representedby “+/−” in FIG. 10A. An individual MFI ratio that is below 30% of theAMFI ratio is represented by “−” in FIG. 10A.

As shown in FIG. 10A, the D60A mutant and the G63A mutant, numberedaccording to SEQ ID NO:41, specifically disrupted or weakened thebinding of pab1876, pab1967, pab1975 and pab1979, but not that of thereference antibody m6C8. The C58A mutant disrupted the binding of allfive antibodies and is likely a structural mutation rather than anepitope-specific one. The C74A mutant had weak expression and could notbe used for binding comparison.

Furthermore, the anti-GITR antibodies 231-32-15, pab1876, and m6C8 werecompared for their binding to wild type versus mutant human GITR.Briefly, wild type human GITR and two GITR alanine mutants (the D60Amutant and the G63A mutant, numbered according to SEQ ID NO:41) wereexpressed on the surface of 1624-5 cells as described above and testedin a flow cytometry analysis as described above where cells were firststained using 2 μg/ml of the monoclonal antibodies 231-32-15, pab1876,and m6C8, or a polyclonal antibody conjugated to APC, and then stainedusing a secondary anti-IgG antibody if necessary for detection (APCconjugated; 1:1000; BD Cat No. 109-136-097). All the mean fluorescenceintensity (MFI) values were calculated as the mean of two measurements.The MFI value of the tested monoclonal antibody for a particular celltype was divided by the MFI value of the polyclonal antibody for thesame cell type, generating a total of nine MFI ratios (monoclonalantibody/polyclonal antibody): MFI ratio_(231-32-15, WT), MFIratio_(pab1876, WT), MFI ratio_(m6C8, WT), ratio_(231-32-15, D60A), MFIratio_(pab1876, D60A), MFI ratio_(m6C8, D60A), MFIratio_(231-32-15, G63A), MFI ratio_(pab1876, G63A), and MFIratio_(m6C8, G63A). The percentage of binding of an antibody to the GITRalanine mutants relative to the wild type GITR was calculated bydividing a particular MFI ratio for the GITR alanine mutants by thecorresponding MFI ratio for the wild type (e.g., dividing MFIratio_(pab1876, D60A) by MFI ratio_(pab1876, WT)). The percentage ofreduction in binding was determined by calculating, e.g., 100%*(1−(MFIratio_(pab1876, D60A)/MFI ratio_(pab1876, WT))).

As shown in FIG. 10B, the D60A mutant and the G63A mutant specificallydisrupted or weakened the binding of 231-32-15 and pab1876, but not thatof m6C8. The percentages shown in FIG. 10B are the percentages of GITRpositive cells in each plot. When tested using the cells expressing GITRD60A, antibody binding was reduced by 82% and 88% for 231-32-15 andpab1876, respectively, compared with a 10% reduction for m6C8.Similarly, when tested using the cells expressing GITR G63A, the bindingof 231-32-15 and pab1876 was reduced by 37% and 59%, respectively,whereas the binding of m6C8 was increased by 62%.

As further evidence for the binding characteristics of the anti-GITRantibodies, the binding of the antibodies to cynomolgus GITR wascompared. The immature protein of cynomolgus GITR comprises the aminoacid sequence of SEQ ID NO:44. To increase protein expression, the firstresidue of the signal peptide of cynomolgus GITR was replaced bymethionine, generating V1M cynomolgus GITR. A mutant cynomolgus GITRV1M/Q62P/S63G, where the amino acid residues at the positions 62 and 63(GlnSer), numbered according to SEQ ID NO:44, were replaced by thecorresponding residues in human GITR (ProGly), was then generated. FIG.11A is a sequence alignment between human GITR, V1M cynomolgus GITR, andV1M/Q62P/S63G cynomolgus GITR. The three proteins shown in FIG. 11A wereexpressed on the surface of 1624-5 cells as described above and testedin a flow cytometry analysis as described above where cells were firststained using 2 μg/ml of the monoclonal antibodies 231-32-15, pab1876,and m6C8, or a polyclonal antibody conjugated to APC, and then stainedusing a secondary anti-IgG antibody (APC conjugated; 1:1000; BD Cat no.109-136-097).

As shown in FIG. 11B, the anti-GITR antibodies 231-32-15 and pab1876displayed binding only to the cells expressing V1M/Q62P/S63G cynomolgusGITR, but not the cells expressing VIM cynomolgus GITR.

8.5 Example 5: Epitope Mapping of Anti-OX40 Antibodies

This example characterizes the epitope of the anti-OX40 antibodiespab1949w, pab2049 and a reference anti-OX40 antibody pab1928. Theantibody pab1928 was generated based on the variable regions of theantibody Hu106-122 provided in U.S. Patent Publication No. US2013/0280275 (herein incorporated by reference). pab1928 comprises aheavy chain of the amino acid sequence of SEQ ID NO:106 and a lightchain of the amino acid sequence of SEQ ID NO:107.

8.5.1 Epitope Mapping—Alanine Scanning

The binding characteristics of pab1949w, pab2049 and the referenceantibody pab1928 were assessed by alanine scanning. Briefly, theQuikChange HT Protein Engineering System from Agilent Technologies(G5901A) was used to generate human OX40 mutants with alaninesubstitutions in the extracellular domain. The human OX40 mutants wereexpressed on the surface of 1624-5 cells using standard techniques oftransfection followed by transduction as described above.

Cells expressing correctly folded human OX40 mutants, as evidenced bybinding to a polyclonal anti-OX40 antibody in flow cytometry, werefurther selected for a sub-population that expressed human OX40 mutantsthat did not bind the monoclonal anti-OX40 antibody pab1949w, pab2049,or pab1928. Cells that exhibited specific antibody binding wereseparated from the non-binding cell population by preparative,high-speed FACS (FACSAriaII, BD Biosciences). Antibody reactive ornon-reactive cell pools were expanded again in tissue culture and, dueto the stable expression phenotype of retrovirally transduced cells,cycles of antibody-directed cell sorting and tissue culture expansionwere repeated, up to the point that a clearly detectable anti-OX40antibody (pab1949w, pab2049, or pab1928) non-reactive cell populationwas obtained. This anti-OX40 antibody non-reactive cell population wassubjected to a final, single-cell sorting step. After several days ofcell expansion, single cell sorted cells were again tested for bindingto a polyclonal anti-OX40 antibody and non-binding to monoclonalantibody pab1949w, pab2049 or pab1928 using flow cytometry. Briefly,1624-5 cells expressing individual human OX40 alanine mutants wereincubated with the monoclonal anti-OX40 antibody pab1949w, pab2049 orpab1928. For each antibody, two antibody concentrations were tested(pab1949w: 2 μg/ml and 0.5 μg/ml; pab2049: 1.8 μg/ml and 0.3 μg/ml;pab1928: 1.1 μg/ml and 0.4 μg/ml). The polyclonal anti-OX40 antibody(AF3388, R&D systems) conjugated with APC was diluted at 1:2000. Fcreceptor block (1:200; BD Cat no. 553142) was added, and the sampleswere incubated for 20 minutes at 4° C. After washing, the cells wereincubated with a secondary anti-IgG antibody if necessary for detection(PE conjugated; BD Cat no. 109-116-097) for 20 min at 4° C. The cellswere then washed and acquired using a flow cytometer (BD Biosciences).

To connect phenotype (polyclonal anti-OX40 antibody +, monoclonalanti-OX40 antibody −) with genotype, sequencing of single cell sortedhuman OX40 mutants was performed. FIG. 12 is a table showing the humanOX40 alanine mutants that still bind the polyclonal anti-OX40 antibodybut do not bind the monoclonal anti-OX40 antibody pab1949w, pab2049, orpab1928. All the residues are numbered according to the mature aminoacid sequence of human OX40 (SEQ ID NO:72). “+” indicates binding and“−” indicates loss of binding based on flow cytometry analysis.

***

The invention is not to be limited in scope by the specific embodimentsdescribed herein. Indeed, various modifications of the invention inaddition to those described will become apparent to those skilled in theart from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims.

All references (e.g., publications or patents or patent applications)cited herein are incorporated herein by reference in their entirety andfor all purposes to the same extent as if each individual reference(e.g., publication or patent or patent application) was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes.

Other embodiments are within the following claims.

1.-188. (canceled)
 189. A method of modulating an immune response in asubject in need thereof, the method comprising administering to thesubject an effective amount of a multispecific antibody comprising afirst antigen-binding region that specifically binds human OX40 and asecond antigen-binding region that specifically binds human GITR,wherein: a. the first antigen-binding region comprises a first heavychain comprising CDRs VH-CDR1, VH-CDR2, and VH-CDR3, and a first lightchain comprising CDRs VL-CDR1, VL-CDR2, and VL-CDR3, wherein the firstheavy chain comprises the VH-CDR1, VH-CDR2, and VH-CDR3 amino acidsequences of the VH amino acid sequence of SEQ ID NO: 54, and the firstlight chain comprises the VL-CDR1, VL-CDR2, and VL-CDR3 amino acidsequences of the VL amino acid sequence of SEQ ID NO: 55 and b. thesecond antigen-binding region comprises a second antibody heavy chaincomprising CDRs VH-CDR1, VH-CDR2, and VH-CDR3 and a second antibodylight chain comprising CDRs VL-CDR1, VL-CDR2, and VL-CDR3, wherein: i.the VH-CDR1 comprises the amino acid sequence of X₁YX₂MX₃ (SEQ IDNO:87), wherein X₁ is D, E or G; X₂ is A or V; and X₃ is Y or H; ii. theVH-CDR2 comprises the amino acid sequence ofX₁IX₂TX₃SGX₄X₅X₆YNQKFX₇X₈(SEQ ID NO:88), wherein X₁ is V or L; X₂ is R,K or Q; X₃ is Y or F; X₄ is D, E or G; X₅ is V or L; X₆ is T or S; X₇ isK, R or Q; and X₈ is D, E or G; iii. the VH-CDR3 comprises the aminoacid sequence of SEQ ID NO:3; iv. the VL-CDR1 comprises the amino acidsequence of KSSQSLLNSX₁NQKNYLX₂ (SEQ ID NO:90), wherein X₁ is G or S;and X₂ is T or S; v. the VL-CDR2 comprises the amino acid sequence ofSEQ ID NO:5; and vi. the VL-CDR3 comprises the amino acid sequence ofQNX₁YSX₂PYT (SEQ ID NO:92), wherein X₁ is D or E; and X₂ is Y, F or S.190. The method of claim 189, wherein: a. the VH-CDR1 of the firstantigen-binding region comprises the amino acid sequence of SEQ IDNO:47; b. the VH-CDR2 of the first antigen-binding region comprises theamino acid sequence of SEQ ID NO:48; c. the VH-CDR3 of the firstantigen-binding region comprises the amino acid sequence of SEQ IDNO:49; d. the VL-CDR1 of the first antigen-binding region comprises theamino acid sequence of SEQ ID NO:50; e. the VL-CDR2 of the firstantigen-binding region comprises the amino acid sequence of SEQ IDNO:51; and f. the VL-CDR3 of the first antigen-binding region comprisesthe amino acid sequence of SEQ ID NO:52 or SEQ ID NO:53.
 191. The methodof claim 189, wherein the first antigen-binding region comprises aVL-CDR3 comprising the amino acid sequence of SEQ ID NO:52.
 192. Themethod of claim 189, wherein the first antigen-binding region comprisesa VL-CDR3 comprising the amino acid sequence of SEQ ID NO:53.
 193. Themethod of claim 189, wherein: a. (i) the VH-CDR1 of the secondantigen-binding region comprises the amino acid sequence of X₁YAMX₂ (SEQID NO:1), wherein X₁ is D, G, or E; and X₂ is Y or H; (ii) the VH-CDR2of the second antigen-binding region comprises the amino acid sequenceof X₁IRTYSGX₂VX₃YNQKFX₄X₅ (SEQ ID NO:2), wherein X₁ is V or L; X₂ is Dor G; X₃ is T or S; X₄ is K, R, or Q; and X₅ is D, E, or G; (iii) theVH-CDR3 of the second antigen-binding region comprises the amino acidsequence of SEQ ID NO:3; (iv) the VL-CDR1 of the second antigen-bindingregion comprises the amino acid sequence of KSSQSLLNSX₁NQKNYLT (SEQ IDNO:4), wherein X₁ is G or S; (v) the VL-CDR2 of the secondantigen-binding region comprises the amino acid sequence of SEQ ID NO:5;and (vi) the VL-CDR3 of the second antigen-binding region comprises theamino acid sequence of QNX₁YSX₂PYT (SEQ ID NO:6), wherein X₁ is D or E;and X₂ is Y or F; b. the VH-CDR1 of the second antigen-binding regioncomprises the amino acid sequence of SEQ ID NO:7, 8, or 9; c. theVH-CDR2 of the second antigen-binding region comprises the amino acidsequence of SEQ ID NO:10, 11, 12, or 13; d. the VL-CDR1 of the secondantigen-binding region comprises the amino acid sequence of SEQ ID NO:14or 15; e. the VL-CDR3 of the second antigen-binding region comprises theamino acid sequence of SEQ ID NO:16 or 17; f. the VH-CDR1, the VH-CDR2,and the VH-CDR3 of the second antigen-binding region comprise the aminoacid sequences of SEQ ID NOs:7, 10, and 3; 8, 11, and 3; 9, 12, and 3;or 9, 13, and 3, respectively; and/or the VL-CDR1, the VL-CDR2, and theVL-CDR3 of the second antigen-binding region comprise the amino acidsequences of SEQ ID NOs:14, 5, and 16; or 15, 5, and 17, respectively;or g. the VH-CDR1, the VH-CDR2, the VH-CDR3, the VL-CDR1, the VL-CDR2,and the VL-CDR3 of the second antigen-binding region comprise the aminoacid sequences of SEQ ID NOs:7, 10, 3, 14, 5, and 16, respectively. 194.The method of claim 189, wherein: a. the VH of the first antigen-bindingregion comprises an amino acid sequence that is at least 75% identicalto the amino acid sequence of SEQ ID NO:54; b. the VH of the firstantigen-binding region comprises the amino acid sequence of SEQ IDNO:54; c. the VH of the first antigen-binding region comprises an aminoacid sequence derived from a human IGHV3-73 germline sequence; d. the VLof the first antigen-binding region comprises an amino acid sequence atleast 75% identical to the amino acid sequence of SEQ ID NO:55 or SEQ IDNO:56; e. the VL of the first antigen-binding region comprises the aminoacid sequence of SEQ ID NO:55 or 56; f. the VL of the firstantigen-binding region comprises an amino acid sequence derived from ahuman IGKV2-28 germline sequence; g. the VH and the VL of the firstantigen-binding region comprise the amino acid sequences of SEQ IDNOs:54 and 55; or 54 and 56, respectively; or h. the VH of the firstantigen-binding region comprises an amino acid sequence derived from ahuman IGHV3-73 germline sequence and a VL comprising an amino acidsequence derived from a human IGKV2-28 germline sequence.
 195. Themethod of claim 189, wherein: a. the VH of the second antigen-bindingregion comprises the amino acid sequence of SEQ ID NO:25; b. the VH ofthe second antigen-binding region comprises an amino acid sequence atleast 75% identical to the amino acid sequence of SEQ ID NO:18, 20, 22,or 24; c. the VH of the second antigen-binding region comprises theamino acid sequence of SEQ ID NO:18, 20, 22, or 24; d. the VH of thesecond antigen-binding region comprises an amino acid sequence derivedfrom a human IGHV1-2 germline sequence; e. the VL of the secondantigen-binding region comprises the amino acid sequence of SEQ IDNO:26; f. the VL of the second antigen-binding region comprises an aminoacid sequence at least 75% identical to the amino acid sequence of SEQID NO:19, 21, or 23; g. the VL of the second antigen-binding regioncomprises the amino acid sequence of SEQ ID NO:19, 21, or 23; h. the VLof the second antigen-binding region comprises an amino acid sequencederived from a human IGKV4-1 germline sequence; i. the VH and the VL ofthe second antigen-binding region comprise the amino acid sequences ofSEQ ID NOs:18 and 19; 20 and 21; 22 and 23; or 24 and 23, respectively;or j. the VH of the second antigen-binding region comprises an aminoacid sequence derived from a human IGHV1-2 germline sequence and a VLcomprising an amino acid sequence derived from a human IGKV4-1 germlinesequence.
 196. The method of claim 189, wherein: a. the first heavychain comprises the amino acid sequence of SEQ ID NO:59, 60, 66, 118,119, or 125; b. the first light chain comprises the amino acid sequenceof SEQ ID NO:67, 68, 69, or 70; c. the first heavy chain comprises theamino acid sequence of SEQ ID NO:59 and the first light chain comprisesthe amino acid sequence of SEQ ID NO:67 or 69; or d. the first heavychain comprises the amino acid sequence of SEQ ID NO:118 and the firstlight chain comprises the amino acid sequence of SEQ ID NO:67 or 69.197. The method of claim 189, wherein: a. the second heavy chaincomprises the amino acid sequence of SEQ ID NO:29, 30, 36, 74, 74, or81; b. the second light chain comprises the amino acid sequence of SEQID NO:37 or 38; c. the second heavy chain comprises the amino acidsequence of SEQ ID NO:29 and the second light chain comprises the aminoacid sequence of SEQ ID NO:37; d. the second heavy chain comprises theamino acid sequence of SEQ ID NO:74 and the second light chain comprisesthe amino acid sequence of SEQ ID NO:37; e. the second heavy chaincomprises the amino acid sequence of SEQ ID NO:30 and the second lightchain comprises the amino acid sequence of SEQ ID NO:37; or f. thesecond heavy chain comprises the amino acid sequence of SEQ ID NO:75 andthe second light chain comprises the amino acid sequence of SEQ IDNO:37.
 198. The method of claim 189, wherein the first heavy chainand/or the second heavy chain comprises a heavy chain constant regionselected from the group consisting of human IgG₁, IgG₂, IgG₃, IgG₄,IgA₁, and IgA₂.
 199. The method of claim 198, wherein the first heavychain and/or the second heavy chain comprises an IgG₁ heavy chainconstant region.
 200. The method of claim 199, wherein: a. the firstheavy chain comprises serine, alanine, and valine at amino acidpositions 366, 368, and 407, respectively, and the second heavy chaincomprises tryptophan at amino acid position 366; or b. the first heavychain comprises tryptophan at amino acid position 366, and the secondheavy chain comprises serine, alanine, and valine at amino acidpositions 366, 368, and 407, respectively, numbered according to the EUnumbering system.
 201. The method of claim 200, wherein the first heavychain and/or the second heavy chain comprises aspartate, leucine, andglutamate at amino acid positions 239, 330, and 332, respectively,numbered according to the EU numbering system.
 202. The method of claim189, wherein the first light chain and/or the second light chaincomprises a human kappa light chain constant region or a human lambdalight chain constant region.
 203. The method of claim 199, wherein: a.the first heavy chain comprises the amino acid sequence of SEQ ID NO:54,and the first light chain comprises the amino acid sequence of SEQ IDNO:67; and b. the second heavy chain comprises the amino acid sequenceof SEQ ID NO:18, and the second light chain comprises the amino acidsequence of SEQ ID NO:37.
 204. The method of claim 189, wherein thefirst heavy chain comprises aspartate, leucine, and glutamate at aminoacid positions 239, 330, and 332, respectively, numbered according tothe EU numbering system.
 205. The method of claim 189, wherein thesecond heavy chain comprises aspartate, leucine, and glutamate at aminoacid positions 239, 330, and 332, respectively, numbered according tothe EU numbering system.
 206. The method of claim 189, wherein the firstheavy chain and the second heavy chain comprise aspartate, leucine, andglutamate at amino acid positions 239, 330, and 332, respectively,numbered according to the EU numbering system.
 207. The method of claim198, wherein the heavy chain constant region comprises the amino acidsequence of SEQ ID NO:
 126. 208. The method of claim 189, wherein theimmune response is enhanced or induced in the subject.
 209. The methodof claim 189, wherein the subject has cancer.
 210. The method of claim209, wherein the cancer is selected from the group consisting ofmelanoma, renal cancer, prostate cancer, colon cancer, and lung cancer.211. The method of claim 209, further comprising administering to thesubject an inhibitor of indoleamine-2,3-dioxygenase (IDO).
 212. Themethod of claim 211, wherein the inhibitor is selected from the groupconsisting of epacadostat, F001287, indoximod, and NLG919.
 213. Themethod of claim 209, further comprising administering to the subject avaccine.
 214. The method of claim 213, wherein the vaccine comprises aheat shock protein peptide complex (HSPPC) comprising a heat shockprotein complexed with an antigenic peptide.
 215. The method of claim214, wherein the heat shock protein is hsp70 or hsc70 and is complexedwith a tumor-associated antigenic peptide.
 216. The method of claim 215,wherein the heat shock protein is gp96 and is complexed with atumor-associated antigenic peptide, wherein the HSPPC is derived from atumor obtained from the subject.
 217. The method of claim 217, furthercomprising administering to the subject a checkpoint targeting agent.218. The method of claim 217, wherein the checkpoint targeting agent isselected from the group consisting of an antagonist anti-PD-1 antibody,an antagonist anti-PD-L1 antibody, an antagonist anti-PD-L2 antibody, anantagonist anti-CTLA-4 antibody, an antagonist anti-TIM-3 antibody, anantagonist anti-LAG-3 antibody, an antagonist anti-CEACAM1 antibody, anagonist anti-GITR antibody, and an agonist anti-OX40 antibody.
 219. Themethod of claim 189, wherein the subject has an infectious disease. 220.The method of claim 189, wherein immune response is reduced orinhibited.
 221. The method of claim 189, wherein the subject has anautoimmune or inflammatory disease or disorder.
 222. The method of claim221, wherein the autoimmune or inflammatory disease or disorder isselected from the group consisting of transplant rejection,graft-versus-host disease, vasculitis, asthma, rheumatoid arthritis,dermatitis, inflammatory bowel disease, uveitis, lupus, colitis,diabetes, multiple sclerosis, and airway inflammation.
 223. The methodof claim 189, wherein the subject is human.